Temporal expression of genes coding for aryl-alkamine-N-acetyltransferase and melatonin receptors in circadian clock tissues: Circadian rhythm dependent role of melatonin in seasonal responses.

We investigated at the transcriptional level the role of daily rhythm in melatonin secretion in seasonal responses in the migratory blackheaded bunting (Emberiza melanocephala), which when exposed to short (SP) and long (LP) photoperiods exhibits distinct seasonal life-history states (LHSs). We reproduced the seasonal LHS by subjecting buntings to SP (8 h light: 16 h darkness, 8 L:16D), which maintained the nonmigratory/ nonbreeding phenotype, and to LP (16 L:8D), which induced the premigratory/ prebreeding, migratory/ breeding and nonmigratory/ postbreeding phenotypes. Plasma melatonin measured at 4 h intervals showed loss of the daily rhythm in the LP-induced premigratory/ prebreeding and migratory/ breeding LHSs. Subsequently, mRNA expression of genes coding for the aryl-alkamine-N-acetyltransferase (AANAT; the rate-liming enzyme of melatonin biosynthesis) and for the receptors for melatonin (Mel1A, Mel1B and Mel1C) was examined in the retina, pineal and hypothalamus; the interacting independent circadian clocks comprising the songbird circadian timing system. Except AANAT that was not amplified in the hypothalamus, we found significant alterations in both, the level and persistence of 24 h rhythm in mRNA expression of all genes, albeit with photoperiod and seasonal differences between three circadian clock tissues. Particularly, 24 h mRNA expression pattern of all genes, except retinal Mel1A, lacked a significant daily rhythm in the LP-induced migratory/ breeding LHS. These results underscore the overall importance of the circadian rhythm in the role of melatonin in photoperiodically-controlled seasonal responses in migratory songbirds.

Melatonin Attenuates Early Brain Injury via the Melatonin Receptor/Sirt1/NF-κB Signaling Pathway Following Subarachnoid Hemorrhage in Mice.

Melatonin (Mel) has been reported to alleviate early brain injury (EBI) following subarachnoid hemorrhage (SAH). The activation of silent information regulator 1 (Sirt1), a histone deacetylase, has been suggested to be beneficial in SAH. However, the precise role of Sirt1 in Mel-mediated protection against EBI following SAH has not been elucidated. The present study aims to evaluate the role of melatonin receptor/Sirt1/nuclear factor-kappa B (NF-κB) in this process. The endovascular perforation SAH model was used in male C57BL/6J mice, and melatonin was administrated intraperitoneally (150 mg/kg). The mortality, SAH grade, neurological score, brain water content, and neuronal apoptosis were evaluated. The expression of Sirt1, acetylated-NF-κB (Ac-NF-κB), Bcl-2, and Bax were detected by western blot. To study the underlying mechanisms, melatonin receptor (MR) antagonist luzindole and Sirt1 small interfering RNA (siRNA) were administrated to different groups. The results suggest that Mel improved the neurological deficits and reduced the brain water content and neuronal apoptosis. In addition, Mel enhanced the expression of Sirt1 and Bcl-2 and decreased the expression of Ac-NF-κB and Bax. However, the protective effects of Mel were abolished by luzindole or Sirt1 siRNA. In conclusion, our results demonstrate that Mel attenuates EBI following SAH via the MR/Sirt1/NF-κB signaling pathway.

Melatonin regulates splenocytes proliferation via IP3-dependent intracellular Ca2+ release in seasonally breeding bird, Perdicula asiatica.

Melatonin plays an important role in the immune regulation of birds. Both endogenous and exogenous melatonin modulates lymphocyte proliferation via its specific membrane receptors, Mel(1a), Mel(1b) and Mel(1c), though the mechanisms behind this process are poorly understood. We investigated the differences in melatonin membrane receptor Mel(1a), Mel(1b) and Mel(1c) expression by western blot and reverse transcription reaction and the in vitro effect of melatonin on the intracellular Ca(2+) concentration ([Ca2+]i) in splenocytes of the Indian Jungle Bush Quail, Perdicula asiatica. We used a non-selective melatonin receptor antagonist for Mel(1a) and Mel(1b), luzindole, and the selective Mel(1b) blocker, 4P-PDOT to check the specific role of melatonin receptor on ([Ca2+]i). The expression of Mel(1a), Mel(1b) and Mel(1c) receptors mRNA and protein was upregulated by melatonin (10(-7) M) with a significant high rise in ([Ca2+]i), which was differentially blocked by supplementation of antagonist, luzindole (10(-7) M) and 4P-PDOT (10(-7) M). Furthermore, we noted in vitro effect of melatonin and 2-aminoethoxydiphenyl borate (2-APB), a cell-permeable antagonist of inositol 1, 4, 5-trisphosphate (IP3) receptor to check the rise in ([Ca2+]i) through the IP3 pathway. Significantly low ([Ca2+]i) was noted in melatonin and 2-APB pretreated splenocytes when compared with splenocytes where 2-APB was absent. Thus, our data suggest that melatonin through its membrane receptor induced the elevation of ([Ca2+]i) via IP(3)-dependent pathway for splenocyte proliferation in P. asiatica.

Overexpression of melatonin membrane receptors increases calcium-binding proteins and protects VSC4.1 motoneurons from glutamate toxicity through multiple mechanisms.

Melatonin has shown particular promise as a neuroprotective agent to prevent motoneuron death in animal models of both amyotrophic lateral sclerosis (ALS) and spinal cord injuries (SCI). However, an understanding of the roles of endogenous melatonin receptors including MT1, MT2, and orphan G-protein receptor 50 (GPR50) in neuroprotection is lacking. To address this deficiency, we utilized plasmids for transfection and overexpression of individual melatonin receptors in the ventral spinal cord 4.1 (VSC4.1) motoneuron cell line. Receptor-mediated cytoprotection following exposure to glutamate at a toxic level (25 µm) was determined by assessing cell viability, apoptosis, and intracellular free Ca(2+) levels. Our findings indicate a novel role for MT1 and MT2 for increasing expression of the calcium-binding proteins calbindin D28K and parvalbumin. Increased levels of calbindin D28K and parvalbumin in VSC4.1 cells overexpressing MT1 and MT2 were associated with cytoprotective effects including inhibition of proapoptotic signaling, downregulation of inflammatory factors, and expression of prosurvival markers. Interestingly, the neuroprotective effects conferred by overexpression of MT1 and/or MT2 were also associated with increases in the estrogen receptor ß (ERß): estrogen receptor α (ERα) ratio and upregulation of angiogenic factors. GPR50 did not exhibit cytoprotective effects. To further confirm the involvement of the melatonin receptors, we silenced both MT1 and MT2 in VSC4.1 cells using RNA interference technology. Knockdown of MT1 and MT2 led to an increase in glutamate toxicity, which was only partially reversed by melatonin treatment. Taken together, our findings suggest that the neuroprotection against glutamate toxicity exhibited by melatonin may depend on MT1 and MT2 but not GPR50.

Melatonin receptors in a pleuronectiform species, Solea senegalensis: Cloning, tissue expression, day-night and seasonal variations.

Melatonin receptors are expressed in neural and peripheral tissues and mediate melatonin actions on the synchronization of circadian and circannual rhythms. In this study we have cloned three melatonin receptor subtypes (MT1, MT2 and Mel1c) in the Senegalese sole and analyzed their central and peripheral tissue distribution. The full-length MT1 (1452 nt), MT2 (1728 nt) and Mel1c (1980 nt) cDNAs encode different proteins of 345, 373, 355 amino acids, respectively. They were mainly expressed in retina, brain and pituitary, but MT1 was also expressed in gill, liver, intestine, kidney, spleen, heart and skin. At peripheral level, MT2 expression was only evident in gill, kidney and skin whereas Mel1c expression was restricted to the muscle and skin. This pattern of expression was not markedly different between sexes or among the times of day analyzed. The real-time quantitative PCR analyses showed that MT1 displayed higher expression at night than during the day in the retina and optic tectum. Seasonal MT1 expression was characterized by higher mRNA levels in spring and autumn equinoxes for the retina, and in winter and summer solstices for the optic tectum. An almost similar expression profile was found for MT2, but differences were less conspicuous. No day-night differences in MT1 and MT2 expression were observed in the pituitary but a seasonal variation was detected, being mRNA levels higher in summer for both receptors. Mel1c expression did not exhibit significant day-night variation in retina and optic tectum but showed seasonal variations, with higher transcript levels in summer (optic tectum) and autumn (retina). Our results suggest that day-night and seasonal variations in melatonin receptor expression could also be mediating circadian and circannual rhythms in sole.

Effect of L-NAME-induced hypertension on melatonin receptors and melatonin levels in the pineal gland and the peripheral organs of rats.

Melatonin plays a role in blood pressure (BP) control. The aim of this study was to determine whether melatonin concentrations and melatonin receptor levels are altered in L-NAME-treated, NO-deficient hypertensive rats. Two groups of male adult Wistar rats were investigated: rats (n=36) treated with NO-synthase inhibitor L-NAME (40 mg kg(-1)) and age-matched controls (n=36). BP was measured weekly by tail-cuff plethysmography. After 4 weeks, L-NAME administration increased BP (178+/-1 vs. control 118+/-1 mm Hg). At the end of treatment, rats were killed in regular 4 h intervals over a 24-h period. Melatonin concentrations in the plasma, pineal gland, heart and kidney and melatonin receptor (MT(1)) density in the aorta were determined. A significant daily rhythm of melatonin concentrations was found in the blood, pineal gland, kidney and heart of both control and hypertensive rats. Peak nighttime pineal melatonin concentrations were higher in L-NAME-treated rats than in controls (3.38+/-0.48 vs. 1.75+/-0.33 ng per pineal gland). No differences between both groups were found in melatonin concentrations in blood, kidney and heart or in the MT(1) receptor density in the aorta. Our results suggest that L-NAME treatment enhances melatonin production in the pineal gland, potentially by decreasing an inhibitory effect of NO on melatonin production in the pineal gland. However, the enhanced pineal melatonin formation was insufficient to increase melatonin concentrations in circulation, heart and kidney of L-NAME-treated rats, indicating an increased use of melatonin in hypertensive animals.

Melatonin: a hormone that modulates pain.

AIMS: Melatonin is a hormone synthesized principally in the pineal gland that has been classically associated with endocrine actions. However, several lines of evidence suggest that melatonin plays a role in pain modulation. This paper reviews the available evidence on melatonin's analgesic effects in animals and human beings. MAIN METHODS: A medline search was performed using the terms "melatonin", "inflammatory pain", "neuropathic pain", "functional pain", "rats", "mice", "human", "receptors", "opioid" and "free radicals" in combinations. KEY FINDINGS: The antinociceptive effect of melatonin has been evaluated in diverse pain models, and several findings show that melatonin receptors modulate pain mechanisms as activation induces an antinociceptive effect at spinal and supraspinal levels under conditions of acute and inflammatory pain. More recently, melatonin induced-antinociception has been extended to neuropathic pain states. This effect agrees with the localization of melatonin receptors in thalamus, hypothalamus, dorsal horn of the spinal cord, spinal trigeminal tract, and trigeminal nucleus. The effects of melatonin result from activation of MT(1) and MT(2) melatonin receptors, which leads to reduced cyclic AMP formation and reduced nociception. In addition, melatonin is able to activate opioid receptors indirectly, to open several K(+) channels and to inhibit expression of 5-lipoxygenase and cyclooxygenase 2. This hormone also inhibits the production of pro-inflammatory cytokines, modulates GABA(A) receptor function and acts as a free radical scavenger. SIGNIFICANCE: Melatonin receptors constitute attractive targets for developing analgesic drugs, and their activation may prove to be a useful strategy to generate analgesics with a novel mechanism of action.

Human placental trophoblasts synthesize melatonin and express its receptors.

Although the role of melatonin on fetal development has been the subject of a number of studies, little is known about the function of melatonin in the placenta. We previously showed that melatonin receptors are expressed and are functional in JEG-3 and BeWo cell lines, both in vitro models of human trophoblast. Local synthesis of melatonin in placenta has been proposed, but the human placenta's ability to synthesize melatonin de novo has never been studied. The purpose of this study was to investigate the expression [reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis] and activity (radiometric assay) of melatonin synthesizing enzymes, and characterize the expression of the melatoninergic receptors in human term villous trophoblast. The results show that arylalkylamine N-acetyltransferase and hydroxyindole O-methyltransferase melatonin synthesizing enzymes are expressed and active in villous trophoblast as well as in JEG-3 and BeWo placental choriocarcinoma cells. In addition, immunohistochemical analysis demonstrated the presence of MT1, MT2, and retinoid-related orphan nuclear receptor alpha melatonin receptor proteins in both villous cytotrophoblast and syncytiotrophoblast (STB) as well as in endothelial cells surrounding the fetal capillaries and in the villous mesenchymal core. RT-PCR and western blot analysis in primary cultures of human term trophoblast confirmed the expression of all three melatonin receptors in villous cytotrophoblast and STB cells. This study demonstrates for the first time a local synthesis of melatonin and expression of its receptors in human trophoblasts and strongly suggests a paracrine, autocrine, and/or intracrine role for this indolamine in placental function and development as well as in protection from oxidative stress.

Diurnal differences in melatonin effect on intracellular Ca2+ concentration in chicken spleen leukocytes in vitro.

Melatonin plays a pleiotropic role in the immune system of mammals and birds. Endogenous and exogenous melatonin modulates lymphocyte proliferation via specific MT(1), MT(2) and Mel(1c) membrane receptors, although the mechanisms behind this process are poorly understood. The diurnal changes in the expression and function of melatonin membrane receptors within the immune system have so far received little attention. We investigated the day/night differences in melatonin membrane receptor mRNA expression in chicken lymphoid organs and cultured splenocytes and examined the in vitro effect of melatonin and 2-iodomelatonin on the intracellular Ca(2+) concentration ([Ca(2+)](i)) in chicken splenocytes. In whole organs, expression of all subtypes of Mel membrane receptors was observed, and the level did not change significantly with the time of day. Interestingly, we observed a significant increase in the expression of the transcripts of all receptor subtypes in cultured splenocytes isolated at night compared with cells obtained during the day. In chicken spleen leukocytes isolated during the day, melatonin and 2-iodomelatonin increased [Ca(2+)](i), with only 2-iodomelatonin being effective in the 'night' cells. Luzindole modulated the [Ca(2+)](i) increase caused by melatonin receptor agonists: it potentiated the stimulatory effect of melatonin during the day, but counteracted that evoked by 2-iodomelatonin at night. The results of this study demonstrate that melatonin can induce changes in [Ca(2+)](i) in chicken spleen leukocytes that should modulate proliferation. The effect of melatonin on [Ca(2+)](i) is less pronounced at night, possibly caused by receptor desensitization.

Function and expression of melatonin receptors on human pancreatic islets.

Melatonin is known to inhibit insulin secretion from rodent beta-cells through interactions with cell-surface MT1 and/or MT2 receptors, but the function of this hormone in human islets of Langerhans is not known. In the current study, melatonin receptor expression by human islets was examined by reverse transcription-polymerase chain reaction (RT-PCR) and the effects of exogenous melatonin on intracellular calcium ([Ca2+]i) levels and islet hormone secretion were determined by single cell microfluorimetry and radioimmunoassay, respectively. RT-PCR amplifications indicated that human islets express mRNAs coding for MT1 and MT2 melatonin receptors, although MT2 mRNA expression was very low. Analysis of MT1 receptor mRNA expression at the single cell level indicated that it was expressed by human islet alpha-cells, but not by beta-cells. Exogenous melatonin stimulated increases in intracellular calcium ([Ca2+]i) in dissociated human islet cells, and stimulated glucagon secretion from perifused human islets. It also stimulated insulin secretion and this was most probably a consequence of glucagon acting in a paracrine fashion to stimulate beta-cells as the MT1 receptor was absent in beta-cells. Melatonin did not decrease 3', 5'-cyclic adenosine monophosphate (cyclic AMP) levels in human islets, but it inhibited cyclic AMP in the mouse insulinoma (MIN6) beta-cell line and it also inhibited glucose-stimulated insulin secretion from MIN6 cells. These data suggest that melatonin has direct stimulatory effects at human islet alpha-cells and that it stimulates insulin secretion as a consequence of elevated glucagon release. This study also indicates that the effects of melatonin are species-specific with primarily an inhibitory role in rodent beta-cells and a stimulatory effect in human islets.

[Comparison of melatonin receptor mRNA expression in bilateral paravertebral muscles in adolescent idiopathic scoliosis].

OBJECTIVE: To study the change of melatonin receptor mRNA expression in paravertebral muscles in adolescent idiopathic scoliosis (AIS), and analyze its relationship with the etiology of AIS. METHODS: Twenty cases with average age of (15.1 +/- 2.2) years and average Cobb angle of 56 degrees +/- 16 degrees , including 10 cases with Cobb angle > 50 degrees and 10 cases with Cobb angle 0.05). In AIS group, the ratio of MT2 mRNA expression on concave side compared with convex side in cases with Cobb angle > 50 degrees and cases with Cobb angle 0.05). The MT1 and MT2 mRNA expression showed no significant difference in control group (P > 0.05). CONCLUSIONS: The melatonin receptor expression in bilateral paravertebral muscles in AIS is asymmetric, which may be a secondary change and has no important role in the pathogenesis of AIS.

Expression of melatoninergic receptors in human placental choriocarcinoma cell lines.

BACKGROUND: Melatonin crosses the placenta and enters the fetal circulation. Moreover, experimental data suggest a possible influence of melatonin on placental function and fetal development in humans. To date, the expression and role of melatonin receptors in human placenta choriocarcinoma cell lines and in human term placental tissues remain to be elucidated. METHODS AND RESULTS: Results from RT-PCR, western blotting and confocal microscopy demonstrated that the MT1, MT2 and RORalpha1 melatonin receptors are expressed in the human term placental tissues and in choriocarcinoma cell lines JEG-3 and BeWo. Furthermore, enzyme-linked immunosorbent assay showed that 6-chloromelatonin (a melatonin agonist) inhibits, in a dose-dependent manner, forskolin-stimulated hCG-beta secretion in JEG-3 (P < 0.001) and BeWo (P < 0.05) cells but had no effect on basal human chorionic gonadotrophin (hCG-beta) levels. This effect of 6-chloromelatonin on forskolin-stimulated HCG-beta secretion was abolished by pertussis toxin (PTX), suggesting that melatonin regulates hCG-beta production by an action involving an inhibitory Gi/o protein. In PTX-treated BeWo cells, 6-chloromelatonin stimulated basal hCG-beta secretion (P < 0.001). CONCLUSION: These results demonstrate, for the first time, the expression of melatonin receptors in human term placental tissues and in choriocarcinoma cells and suggest a possible paracrine/autocrine function for melatonin in human placenta.

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.

Melatonin as a local regulator of human placental function.

Melatonin plays a critical role in a variety of mammalian reproductive processes not only acting on the central nervous system but also behaving as a peripheral physiologic regulator. To address the relevance of melatonin to the maintenance of pregnancy at the feto-maternal interface, we investigated the expression of two types of membrane melatonin receptors, MT1 and MT2, as well as arylalkylamine N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT), the two enzymes required for the conversion of serotonin to melatonin, in the human placenta and the effect of melatonin on the release of human chorionic gonadotropin (hCG) from cultured human trophoblast cells. RT-PCR analysis and DNA sequencing revealed that transcripts of MT1, MT2, AA-NAT, and HIOMT were present in the first-trimester human placenta. We also found that melatonin significantly potentiated hCG secretion at optimal concentrations. These results suggest that melatonin may regulate human placental function in a paracrine/autocrine manner, providing evidence for a novel role in human reproduction.

Embryonic and post-embryonic expression of arylalkylamine N-acetyltransferase and melatonin receptor genes in the eye and brain of chum salmon (Oncorhynchus keta).

Melatonin and arylalkylamine N-acetyltransferase (AANAT), the rate-limiting enzyme in melatonin synthesis, have taken on special importance in vertebrate circadian biology. Recent identification of genes encoding two AANAT (AANAT(1) and AANAT(2)) and two subtypes of melatonin receptor (Mel-R; Mel(1a) and Mel(1b)) in several fish species has led to rapid advances in characterizing the physiological roles of melatonin. In the present study, partial cDNAs encoding these four genes were cloned from the eye and brain of chum salmon (Oncorhynchus keta). Based on the nucleotide sequences, we developed highly sensitive real-time PCR systems for these four mRNAs. The development of daily rhythmicity in AANAT(1), AANAT(2), Mel(1a), and Mel(1b) transcript levels was examined in the eye and brain of chum salmon during embryonic and post-embryonic stages (from day -9 to day +180). In a parallel experiment, ocular and brain melatonin levels were measured by radioimmunoassay. Parallelism in developmental changes and in circadian rhythms of AANAT mRNAs and melatonin levels in the eye and the brain supports a hypothesis that the developmental increases of nocturnal melatonin levels results partly from the elevated transcription of AANAT genes. Moreover, abundant expression of AANAT and Mel-R mRNAs in the optic tectum, thalamus, hypothalamus, cerebellum, and eye indicates possible roles of melatonin in visual processing and neuroendocrine regulation, through which melatonin might be involved in migratory behavior of chum salmon.

Immortalized cells from the rat suprachiasmatic nucleus express functional melatonin receptors.

Immortalized SCN2.2 cells retain most biochemical and biophysical characteristics of the native rat SCN including the expression of clock genes and circadian regulatory proteins, and its distinctive pacemaker function. This study assessed the expression and signaling of MT(1) and MT(2) melatonin receptors in SCN2.2 cells. SCN2.2 cells express MT(1) and MT(2) receptors mRNA as detected by RT-PCR. In situ hybridization with digoxigenin-labeled probes demonstrated that mRNA for MT(1) and MT(2) melatonin receptors is expressed mostly in cells with neuronal-like morphology, representing 10.8+/-2.2% and 9.8+/-0.2%, respectively, of the SCN2.2 cell population. MT(1) and MT(2) melatonin receptor proteins are expressed in both rat SCN2.2 cells and rat SCN tissue as demonstrated by Western blot analysis with specific receptor antiserum. Melatonin (0.1-100 nM) inhibited forskolin (20 microM)-stimulated cAMP formation in a dose-dependent manner and this effect was blocked by the competitive melatonin receptor antagonist luzindole (100-1000 nM). Furthermore, melatonin (1 nM) stimulated protein kinase C (PKC) activity by approximately 2-fold. The selective MT(2) receptor antagonist 4P-PDOT (100 nM) blocked this effect, indicating that the melatonin-mediated increase in PKC activity occurs through activation of MT(2) melatonin receptors. We conclude that SCN2.2 cells express functional melatonin receptors, providing an in vitro model to unveil the melatonin signaling pathway(s) involved in the regulation of circadian rhythms.

Expression of membrane and nuclear melatonin receptor mRNA and protein in the mouse immune system.

The neurohormone melatonin plays a fundamental role in neuroimmunomodulation of several mammalian species, including mice. This effect is supported by the existence of specific melatonin-binding sites in murine immunocompetent organs. Moreover, using melatonin receptor analogues, several effects of the neurohormone on mice physiology through its membrane and nuclear receptors have been described. The expression of these receptors has never been studied, despite indirect evidence showing the presence of melatonin receptor in the murine immune system. At present, the MT1 and MT2 membrane receptors, and nuclear receptors belonging to the RZR/ROR family have been related to the immunomodulator effect of melatonin. Here, we show the presence of membrane and nuclear melatonin-binding sites in mouse thymus and spleen, using the specific melatonin membrane (S 20098) and nuclear (CGP 52608) receptor agonist. To confirm the presence of melatonin receptors, we analyzed the presence of membrane and nuclear receptor mRNA and protein by RT-PCR, Southern blot, and Western blot. Thus, we show that MT1 and RORalpha receptor mRNA and protein are expressed in both thymus and spleen, while MT2 receptor mRNA is only detected in the thymus. This expression of melatonin receptors strongly supports the idea of an immunomodulatory role of melatonin through its receptors.