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SARS-CoV-2 has a zoonotic origin and was transmitted to humans via an undetermined intermediate host, leading to infections in humans and other mammals. To enter host cells, the viral spike protein (S-protein) binds to its receptor, ACE2, and is then processed by TMPRSS2. Whilst receptor binding contributes to the viral host range, S-protein:ACE2 complexes from other animals have not been investigated widely. To predict infection risks, we modelled S-protein:ACE2 complexes from 215 vertebrate species, calculated changes in the energy of the complex caused by mutations in each species, relative to human ACE2, and correlated these changes with COVID-19 infection data. We also analysed structural interactions to better understand the key residues contributing to affinity. We predict that mutations are more detrimental in ACE2 than TMPRSS2. Finally, we demonstrate phylogenetically that human SARS-CoV-2 strains have been isolated in animals. Our results suggest that SARS-CoV-2 can infect a broad range of mammals, but few fish, birds or reptiles. Susceptible animals could serve as reservoirs of the virus, necessitating careful ongoing animal management and surveillance.

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In mammals, the nocturnal rise in pineal melatonin is regulated by signals from the endogenous clock, the hypothalamic suprachiasmatic nuclei. There have been few reports on whether anaesthetics which modulate multisynaptic neuronal functions affect melatonin secretion. We studied the effects of three commonly used anaesthetics, halothane, pentobarbital and ketamine, on serum melatonin levels in male New Zealand white rabbits. Seven blood samples were collected, 30-60 min apart, before, during and after anaesthesia. Experiments were performed in the late light and early dark period, so that changes in melatonin secretion would be reflected in the onset and/or level of nocturnal serum melatonin. Serum melatonin levels were determined by radioimmunoassay. Our results indicated that halothane attenuated the release of melatonin and pentobarbital had no apparent effect, whereas ketamine potentiated the release of melatonin. These findings suggest that melatonin levels may be affected in patients anaesthetized with halothane or ketamine, resulting in disturbed biological rhythms, especially the sleep-wake cycle following recovery.

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Twenty-three ICR mice were force fed orally with American ginseng extract, Panax quinquefolius, (Cold FX) for 4 days. Another 20 mice were fed with water as placebo in a similar fashion. Formalin tests which yield typically two phases of pain behavior were done in both groups. Although there was no difference in the first phase between groups, mice treated with Cold FX spent significantly less time in licking and biting of the injured paws in the second phase. The data indicate that American ginseng may have analgesic effect in this chronic pain model.

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The family of melatonin receptors is composed of the mt1, MT2, and Mel1c subtypes. The Mel1c is further divided into one long and two short isoforms. A recent study has shown that, unlike mt1 and MT2, the long form of Mel1c is incapable of activating the pertussis toxin-insensitive G16. Here we used three well-characterized Galphaq chimeras to explore the coupling specificity of the melatonin receptors. The qi5, qo5, and qz5 chimeras can link numerous Gi-coupled receptors to the stimulation of phosphoinositide-specific phospholipase C. Both mt1 and MT2 receptors interacted productively with the Galphaq chimeras, while the long form of Mel1c was totally ineffective. Among the Galphaq chimeras, qo5 was less efficiently coupled to the melatonin receptors. Such differential coupling is best explained by structural differences between the melatonin receptors as well as among the Galphaq chimeras. Since the long form of Mel1c receptor possesses an exceptionally large C-terminal tail, we tested the ability of four melatonin receptor C-terminal tail chimeras (Chi 1-4) to interact with the Galphaq chimeras. The presence of the large C-terminal tail of Mel1c in Chi 1 and Chi 3 markedly hindered their coupling to the Galphaq chimeras. On the other hand, the attachment of either the mtl or MT2 C-terminal tail to a Mel1c backbone produced chimeras (Chi 2 and Chi 4) that were capable of activating the Galphaq chimeras. These findings suggest the involvement of C-terminal regions of melatonin receptors in the recognition of G proteins.

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The possible analgesic effect of melatonin was investigated in young male ICR mice. The formalin test which elicits typically 2 phases of pain response, the acute (first) phase and tonic (second) phase, was used. The test was performed in the late light period when the mice have been reported to be more sensitive to pain. Compared to control mice, no significant difference in nociceptive response was observed when melatonin was injected intraperitoneally at doses of 0.1, 5, and 20, mg/kg body weight. The combined effects of melatonin with diazepam and/or morphine, were also investigated. Melatonin, injected at 20 mg/kg 15 min before formalin test, significantly increased the antinociceptive response of diazepam (1 mg/kg) or morphine (5 mg/kg) in the second phase. In addition, when melatonin was given at 20 mg/kg together with diazepam and morphine, antinociceptive responses in both the first and second phase were increased. These data indicate the synergistic analgesia effect of melatonin with morphine and diazepam and suggest the possible involvement of melatonin as an adjunct medicine for pain patients.

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OBJECTIVE: To examine the inhibitory effect of melatonin (MLT) on the development of pituitary prolactin-producing tumors (prolactinoma) induced by 17-beta-estradiol (E2), in vivo, and explore MLT's oncostatic mechanisms. METHODS: The prolactinomas were established by implanting E2-laden silastic capsules subcutaneously in Sprague-Dawley male rats. MLT doses 0.05, 0.25, 0.50, 1.00, and 2.00 mg/rat were administrated separately to 5 groups subcutaneously starting seven days prior to tumor induction for 97 days. The matched controls were given equal volumes of 4% alcohol in saline. RESULTS: (1) The prolactinoma weights in 0.05, 0.25, 0.50, 1.00 and 2.00 mg MLT dose groups were 25.91% (P > 0.05), 48.78% (P < 0.01), 36.78% (P < 0.05), 31.04% (P > 0.05) and 35.22% (P > 0.05) respectively which were lower than that of control group; (2) The PRL mRNA levels of prolactinoma in 0.05, 0.25, and 0.50 mg MLT dose groups were 33.67% (P < 0.05), 25.51% (P < 0.05) and 41.84% (P < 0.01) respectively which were lower than that of control group as estimated by Northern Blot, and the in situ hybridization studies; (3) The DNA contents of prolactinoma in 0.05, 0.25 and 0.50 mg MLT dose groups were 40.73% (P < 0.001), 51.15% (P < 0.001) and 60.23% (P < 0.001) respectively which were lower than that of control group by laser scanning confocal microscopy; (4) Plasma peroxidative lipid contents in 0.05, 0.25, 0.50, 1.00 and 2.00 mg MLT dose groups were 26.45% (P < 0.05), 23.97% (P < 0.05), 47.11% (P < 0.001), 66.12%(P < 0.001) and 64.46% (P < 0.001) respectively which were lower than that of control group. The correlation coefficient between MLT doses and plasma peroxidative lipid contents was -0.8257 (P < 0.05). CONCLUSIONS: MLT in suitable doses is able to inhibit the development of E2-induced prolactinoma by inhibiting the expression of PRL gene and the DNA synthesis. The link between MLT antioxidative action and its inhibitory effect on development of prolactinoma should be further investigated.

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Prolactinoma is the most common type of primary pituitary tumors. It occurs more frequently in women than in men. Dopaminergic agonists are effective in the shrinkage of prolactin-secreting pituitary tumor and are preferred in some patients. However, pituitary radiotherapy may enable the long-term removal of prolactin-secreting tumor cells. Recent evidence suggests that prolactinoma is a heterogeneous disorder with complicated and multifactorial etiology and pathogenesis. Apparently, a thorough understanding of prolactinoma tumorigenesis would be important. To facilitate investigations on tumorigenesis of prolactinoma, animal models for prolactinomas have been developed. These models have expedited our progress in the recent years. Many researchers consider the F(344) rat to be the most sensitive strain of rats to estrogen (E(2))-induced prolactinoma formation. Nonetheless, E(2) treatment for 60 days also induces the formation of pituitary prolactin-secreting adenoma in male Sprague-Dawley (SD) rats. Evidently, the SD rat is also a good animal for prolactinoma investigations. Following E(2) implantation, prolactinomas developed in the eutopic adenohypophysis in situ and/or ectopic pituitary grafted under the renal capsule in SD rats. These observations favor the hypothesis that prolactinoma growth is the result of pathological changes in the adenohypophysis and/or hypothalamus. In the latter case, abnormal release of hypothalamic dopamine, GABA, or brain-gut peptides (such as cholecystokinin, vasoactive intestinal polypeptide, galanin, angiotensin, opioid peptide, gastrin, gastrin-releasing peptide, pancreatic polypeptide, and adrenocorticotropic hormone) results in some of the pathological changes that may lead to hyperprolactinemia and/or prolactinoma development. Dysregulation of prolactin synthesis and secretion may be the result of prolactin gene modulation. In E(2)-induced rat prolactinomas, prolactin mRNA contents and the expression of some proto-oncogenes, e.g. c-myc and c-ras, TGFalpha and TGFbeta1 mRNA were significantly changed. The above findings are consistent with results in human prolactinoma development. In addition, in rats abnormal expression of the prolactin gene was correlated with hypomethylated status of CpG sites in exons 1, 2 and 4 of the prolactin gene, as well as the increase in hypersensitive sites to DNase 1 in the encoding region of the prolactin gene. In E(2)-treated rats, a point mutation with a base substitution from cytidine (C) to adenine (A) was found at the -36-bp site of the proximal promoter of the prolactin gene in eutopic pituitary prolactinomas, but no change was observed in the same sequence of the prolactin gene in ectopic prolactinoma. The association of a base substitution with the hyperexpression of the prolactin gene in eutopic prolactinomas suggests that different mechanisms may mediate the formation of eutopic and ectopic prolactin-secreting tumors. Melatonin decreases the expression of the prolactin gene in vitro suggesting that this pineal hormone may be a potential anticarcinogen in vivo. It has also been shown that MT(2) (Mel(1b)) melatonin receptors are expressed in anterior pituitary cells. The use of melatonin as a preventive or therapeutic drug for prolactinomas should be further investigated. In summary, improved knowledge on tumorigenesis of prolactinomas, especially in the rat model, was noted. These E(2)-induced rat prolactinoma models would facilitate future investigations, and expected results shall be fruitful and exciting for the development of future drug designs for the prevention and/or treatment of prolactin-secreting pituitary tumors.

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Melatonin, the pineal neurohormone, is an evolutionarily conserved photoperiodic signaling molecule with diverse functions that include the entrainment of human circadian rhythms. Although evidence supporting a direct inhibitory action of melatonin on human cancer cell proliferation exists in the literature, the molecular and cellular signaling mechanisms involved are largely undefined. In our study, significant inhibition of human choriocarcinoma JAr cell proliferation at physiological and pharmacological concentrations of melatonin was observed. 2-Iodomelatonin, a high affinity melatonin receptor agonist, was more potent than melatonin in inhibiting JAr cell proliferation. In addition, the presence of putative melatonin receptors in choriocarcinoma was suggested by the demonstration of specific 2-[125I]iodomelatonin binding to the tumor. Interestingly, the selective MT2 melatonin receptor ligand, 4-phenyl-2-propionamidotetraline (4-P-PDOT), was found to exert not only concentration-dependent anti-proliferative actions on JAr cells, but also additive effects with melatonin in inhibiting JAr cell proliferation. Furthermore, MT2 melatonin receptor gene expression by JAr cells was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization (ISH). Taken together, our data suggest that the reported anti-proliferative action of melatonin on human choriocarcinoma JAr cells may be mediated, in part, by MT2 melatonin receptor. Moreover, analysis of melatonin effect on cell cycle kinetics indicated that G1/S transition delay may underlie the observed inhibition of choriocarcinoma cell proliferation by melatonin.

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The circadian melatonin rhythm with high levels in the dark period is important for the synchronization of reproductive response to appropriate environmental conditions in animals. The target sites of melatonin action on reproductive functions remain to be clarified. Using autoradiography (ARG) and radioreceptor binding assays with 2[125I]iodomelatonin, a melatonin agonist, as the radioligand, studies on the sites of melatonin action have increased significantly in the last ten years. The recent cloning of melatonin receptor subtypes also allowed the characterization of receptor(s) to the molecular level. Earlier reports have documented that the hypothalamic-pituitary axis plays a vital role in the regulation of reproduction by melatonin. This is supported in part by the demonstration of melatonin receptors in the suprachiasmatic nuclei (SCN) in the brain and pars tuberalis (PT) in the pituitary. However, the nature of SCN and PT involvement in the reproductive action of melatonin remains unknown. In addition to the hypothalamus and pituitary, the two classical sites of melatonin action, other targets have been identified. The recent demonstration of 2[125I]iodomelatonin binding sites or melatonin receptors in the testis, epididymis, vas deferens, prostate, ovary and mammary gland suggest the concept of multiple sites of melatonin action on the reproductive system. The presence of melatonin receptors in the said tissues is consistent with earlier reports of direct melatonin actions on different levels of the reproductive system. This multiple levels of melatonin action, from the hypothalamus, pituitary, gonads to other reproductive tissues form a robust system of photoperiodic control in animal reproduction. This would guarantee successful gestation and delivery of the offspring at a time with optimum food availability and ultimately favourable for the survival of species. Molecular and cellular studies of melatonin signaling system(s), its regulation and effects on downstream functional events in the future may provide new insights and directions for the study of the physiology and pharmacology of fertility and contraception in animals and humans.

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Secretion of pineal melatonin exhibits a diumal rhythm and a seasonal rhythm in humans. Night-time melatonin is high at 3-5 year-old and decreases with age. Many drugs and pathological conditions also change melatonin levels in the circulation. Melatonin has a mild sedative effect and has been used effectively in synchronizing the sleep-wake cycle of patients with sleep disorders. Immunoenhancing, anti-cancer, anti-aging and anti-oxidant effects of melatonin have been proposed. Recent studies suggest that melatonin receptors are present in central and peripheral tissues. The importance of melatonin receptors on the nervous, reproductive, immune and renal functions is implicated. Studies on the molecular biology, physiology and pathology of melatonin receptors in different tissues are progressing rapidly. The physiological and pathological changes in melatonin secretion, multifarious melatonin actions, and diverse melatonin receptors reported suggest that melatonin is a photoperiodic signal with clinical significance in humans.

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We have compared the 50% inhibition values of 2-[125I]iodomelatonin ([125I]Mel) competition curves by melatonin and 3 naphthalenic ligands, N-[2-(7-methoxy-1-naphthyl) ethyl] cyclobutane carboxamide (S20642), N-propyl N-[2-(7-methoxy-1-naphtyl) ethyl] urea (S20753), and N-[2-(7-methoxy-1-naphthyl) ethyl] crotonamide (S20750), using membrane preparations of four tissues (lung, spleen, brain, and kidney) of the chicken simultaneously. In retired breeders, we have demonstrated that the affinities of S20642 were similar in the lung and spleen. However they were 2-fold lower in the brain and 80-fold lower in the kidney. Similar differential binding affinities to the melatonin receptors were observed in the four tissues of young male chicks. This suggests that age and sex have little influence on the differential inhibitory properties of melatonin and S20642 in the tissues studied. The addition of guanosine 5'-O-thiotriphosphate (GTPgammaS), which encouraged the uncoupling of melatonin receptor to the G protein complex, lowered the binding affinity of melatonin and S20642 in the tissues studied but their differential affinities in the four tissues were however maintained. The affinities of 5-methoxy-N-cyclopropanoyltryptamine (CPMT) in the kidney were also 5-10-fold lower than those in the lung, spleen, and brain of young male chicks. The distinctive differential affinities of melatonin, S20642, and CPMT for [125I]Mel binding sites in the chicken lung, spleen, brain, and kidney indicated that the binding sites in these tissues are heterogeneous. Our study implicated that the naphthalenic ligand S20642 may be a useful melatonin analog to distinguish melatonin receptor subtypes in tissues and a possible drug candidate worthwhile for further investigations.

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Effects of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and cations on 2-[125I]iodomelatonin binding were investigated in membrane preparations of the chicken spinal cord. At concentrations of 10 and 50 mumol/l, GTP gamma S dose-dependently increased (p < 0.05) the equilibrium dissociation constant (Kd) and depressed (p < 0.05) the maximum number of binding sites (Bmax). Na+ at a concentration of 125 mmol/l significantly increased (p < 0.05) the Kd and decreased (p < 0.05) the Bmax, and Mg2+ (2.5 mmol/l) significantly increased (p < 0.05) the Bmax without changes in Kd. In addition, Na+ and Mg2+ affected the interactions of GTP gamma S with melatonin receptors. In the spinal cord explants, melatonin (10 nmol/l) attenuated forskolin-stimulated cyclic AMP production by 53.1%, and preincubation with pertussis toxin abolished this effect of melatonin. These results suggest that the melatonin receptors in the chicken spinal cord are linked to its second messenger via a pertussis-toxin-sensitive guanine-nucleotide-binding protein, and that cations modulate these receptors. Our studies further support a previous hypothesis that melatonin exerts a direct action on spinal cord functions.

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The effect of pinealectomy on the characteristics of melatonin receptors in the chicken kidney was studied. One-day-old chicks were operated and kept under a 12 h/12 h light/dark photoperiod. Six weeks after operation, the animals were sacrificed at mid-light and mid-dark. Serum melatonin was determined by radioimmunoassay and kidney melatonin receptors were studied by radioreceptor assay using the melatonin agonist 2-[125I]iodomelatonin as the radioligand. Pinealectomy significantly reduced the mid-dark serum melatonin level and abolished the diurnal rhythm of 2-[125I]-iodomelatonin binding in the kidney. The density of 2-[125I]-Iodomelatonin binding sites in the kidney at mid-dark was increased significantly to a value comparable to the mid-light density after pineal ablation. Our results suggest that melatonin receptors in the chicken kidney are directly regulated by melatonin in the circulation. The coupling of kidney melatonin receptors to adenylate cyclase was investigated. The basal and forskolin-stimulated cAMP production in chicken kidney explants was studied following melatonin or melatonin plus pertussis toxin treatment. Levels of cAMP in chicken kidney explants were extracted and determined by radioimmunoassay. Melatonin had no effect on basal cAMP levels. However, melatonin significantly inhibited the forskolin-stimulated cAMP accumulation at a concentration of 10 pmol/l. Inhibitory effects of melatonin on the forskolin-stimulated cAMP increase in the chicken kidney were totally blocked by preincubating the kidney tissue with 1.0 micrograms/ml pertussis toxin. Our results suggest that kidney melatonin receptors may modulate the adenylate cyclase leading to biological responses in the renal system.

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To investigate whether melatonin has a direct action on the cardiovascular system, putative melatonin receptors were studied in quail heart membrane preparations using the specific melatonin agonist 2-[125I]iodomelatonin (125I]Mel, as the radioligand. The [125I]mel binding demonstrated in the mature quail heart was saturable, highly 5.2 pM; Bmax = 1.32 +/- 0.25 fmol/mg protein; n = 8). The linear Scatchard plots and the close to unity Hill coefficient indicated a single class of binding sites. The pharmacological profile was in the affinity order of 2-iodomelatonin = 2-phenylmelatonin > melatonin > 6-chloromelatonin >> 6-hydroxymelatonin > 6-sulphatoxymelatonin >> N-acetylserotonin>>>5-hydroxytryptamine. Guanosine 5'-triphosphate and guanosine 5'-O-(3-thiotriphosphate) (GTP gammaS) dose dependently inhibited the binding. Ten microM GTPgammaS lowered the binding affinity by 50% in saturation studies. The order of potency of inhibition by cations was: Ca2+ > Mg2+ > Li+ > Na+ > K+ > choline chloride. Contrary to most other melatonin binding sites, millimolar concentrations of Ca2+ and Mg2+ did not promote binding in the quail heart membranes. In vitro autoradiography indicated homogenous labeling in the heart. Our results demonstrated [125I]Mel binding sites in the quail heart. That guanine nucleotides and Na+ inhibited the binding indicated that these putative melatonin receptors are coupled to guanine nucleotide-binding proteins (G-proteins).

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We have compared the pharmacological characteristics of 2-[125I]iodomelatonin binding to crude membrane preparations of the lung, spleen, brain and kidney of chicken. Saturation studies indicated significant differences (p < 0.05) in the equilibrium dissociation constant (Kd) and maximum number of binding site (Bmax) values among the four tissues studied. The descending order of affinities was lung = spleen > brain > kidney. Competition curves of 2-[125I]iodomelatonin binding to crude membrane preparations of all four chicken tissues by melatonin were studied simultaneously to reduce individual, physiological, age and interassay variations. Similar competition experiments were also performed on 2-phenylmelatonin, 2-iodomelatonin, 6-chloromelatonin, 6-hydroxymelatonin and N-acetylserotonin (NAS). Concentrations of indoles which inhibited 50% of specific 2-[125I]iodomelatonin binding (IC50) were calculated. The IC50 of 2-[125I]iodomelatonin inhibition curves by the indole compounds in different tissues showed the following descending orders of affinity: (1) melatonin: lung = spleen > brain > kidney, (2) 2-phenylmelatonin: lung = spleen = brain = kidney, (3) 2-iodomelatonin: lung = spleen = kidney > brain, (4) 6-chloromelatonin: lung = spleen = kidney > brain, (5) 6-hydroxymelatonin: kidney > lung = spleen = brain, and (6) NAS: kidney > lung = spleen > brain. The non-hydrolizable GTP analog, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), exhibited differential effects on the saturable binding of the four tissues. GTP gamma S increased the Kd of 2-[125I]iodomelatonin binding by 2- to 3-fold in the lung and spleen, 0.5-fold in the brain and 1-fold in the kidney. Based on our findings, we would like to suggest that the 2-[125I]iodomelatonin binding sites in these four tissues may belong to three different high affinity (picomolar) subtypes of melatonin receptor. We name them cML1A represented by the lung and spleen, cML1B by the brain and cML1C by the kidney.

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[125I]-Labelled iodomelatonin binding to brain membrane preparations was examined in adults of 4 species of salmonid fishes; 3 species were of cultured stock (Arctic charr, Atlantic salmon, rainbow trout) and 1 was taken from the wild (coho salmon). The specific binding of 2-[125I]iodomelatonin to the brain membrane preparations collected from all 4 species was specific, stable, saturable, reversible and of high affinity. Competitive inhibition studies showed that only melatonin, 2-iodomelatonin, 6-chloromelatonin and N-acetylserotonin showed significant inhibition of radioligand binding. In the 4 species studied, the equilibrium dissociation constant (Kd) ranged from 9.3 +/- 0.2 pmol/l in coho salmon to 13.2 +/- 0.7 pmol/l in rainbow trout, and those in rainbow trout were significantly higher (p < 0.05) than in the others. The maximum number of binding sites (Bmax) of the Atlantic salmon preparations (14.3 +/- 0.7 fmol/mg protein) was significantly higher than those of the other 3 species (means ranging from 9.9 to 11.6 fmol/mg protein). Hill coefficients of all 4 species were close to unity indicating one class of binding site. The interspecies differences in Kd and Bmax could not be correlated with phylogeny or life history of the various species. For the three cultured species, samples were collected at both mid-dark and mid-light to examine for diurnal variations; none were found.

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Pineal melatonin modulates the mammalian immune system. In vivo studies showed that melatonin enhanced the natural and acquired immunity while in vitro studies demonstrated its inhibitory influence. The mechanism of melatonin action on the immune system remains unknown. Actions through lymphokines or opioid release or via other endocrine changes have been proposed. In this paper, a direct action of melatonin on the lymphoid tissue is hypothesized. 2-[125I]Iodomelatonin binding sites have been identified in the membrane homogenates of thymus, bursa of Fabricius and spleens of a number of birds and mammals. The bindings were stable, saturable, reversible, specific and of high affinity. The Bmax ranged from 0.6 to 3.9 fmol/mg protein. The Kd was in the physiological range of circulating melatonin levels, about 30-70 pmol/l. The binding sites in the primary lymphoid organs demonstrated diurnal variation in density, with higher levels found at the middle of the light period. However, those in the spleen did not vary with the time of the day. An age-dependent decrease in the density was also found in the chicken bursa of Fabricius. In addition, when the nocturnal melatonin secretion was suppressed by constant light exposure, the density of the binding sites increased in the guinea pig spleen. Immunosuppression with cortisol injection in young ducks decreased the density of the melatonin binding sites in the thymus. The regulation of the binding characteristics by physiological variation in melatonin levels and/or immunological status of the animals provide evidence that these 2-[125I]iodomelatonin binding sites in the lymphoid tissues may be physiologically significant and represent true melatonin receptors. The melatonin receptors in the lymphoid organs may be coupled to a G protein as Guanosine 5'-0-(3-thiotriphosphate inhibited 2-[125I]iodomelatonin binding in the spleen by increasing the Kd and decreasing the Bmax.

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The existence of melatonin receptors in adrenal glands is suggested by effects on rodent adrenal function which follow pinealectomy or in vivo and in vitro melatonin treatment. In 1992, Persengiev and coworkers reported specific binding of 2-[125I]iodomelatonin to rat adrenal tissue. In order to study adrenal binding further we have done binding studies on adrenal membranes from ducks sacrificed at midlight. We observed binding of 2-[125I]iodomelatonin which was specific, rapid, saturable, stable, reversible and of high affinity. Scatchard plots were linear and Hill coefficients were close to unity supporting the existence of a single class of 2-[125I]iodomelatonin binding sites. On Scatchard analysis the Kd was in the physiological range (27.4 pmol/l) together with a Bmax of 3.38 fmol/mg protein. Sites were highly specific to melatonin and its two synthetic analogs, 2-iodomelatonin and 6-chloromelatonin in pharmacological studies. In autoradiographic studies on the chicken adrenal gland, one class of 2-[125I]iodomelatonin binding site was demonstrated with a Kd of 58.8 pmol/l and Bmax of 182 fmol/g tissue. Kinetic and pharmacological studies indicated that these sites are saturable, reversible, and of high specificity and affinity. These findings in the chicken are similar to those of the duck adrenal data obtained by radioreceptor assay. In preliminary studies, no significant difference was found between 2-[125I]iodomelatonin binding to the adrenal glands collected at midlight and middark. These high-affinity binding sites are similar to those reported in a wide variety of tissues and are consistent with the hypothesis of direct action of melatonin on the adrenal gland.(ABSTRACT TRUNCATED AT 250 WORDS)

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Using 2-[125I]iodomelatonin as the radioligand, putative melatonin receptors in the duck adrenal gland were investigated. 2-[125I]Iodomelatonin binding to the membrane preparations of duck adrenals collected at mid-light was specific, rapid, saturable, stable, reversible and of high affinity. Scatchard analyses showed one class of binding sites with an equilibrium dissociation constant of 27.4 +/- 2.9 pmol/l and a maximum number of binding sites of 3.38 +/- 0.26 fmol/mg protein. Binding of 2-[125I]iodomelatonin in different subcellular fractions demonstrated the following descending order of density: mitochondrial > nuclear > microsomal >>> cytosol. Pharmacological studies indicated that these sites were highly specific to melatonin. As 2-[125I]iodomelatonin is a specific agonist of melatonin, it is proposed that the sites studied are adrenal melatonin receptors.

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