Melatonin Target Research Articles

Neutrophils as a specific target for melatonin and kynuramines: effects on cytokine release

A growing body of evidence suggests that the pineal hormone, melatonin, has immunomodulatory properties, although very little is known about its effect on leukocytes. Therefore, we aimed to investigate the effect of melatonin and its oxidation product N 1-acetyl- N 2-formyl-5-methoxykynuramine (AFMK) on cytokine production by neutrophils and peripheral blood mononuclear cells (PBMCs). AFMK (0.001–1 mM) inhibits the lipopolysaccharide (LPS)-mediated production of tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) more efficiently in neutrophils than PBMCs. Moreover, the inhibitory activity of AFMK is stronger than that of melatonin. Interestingly, monocytes efficiently oxidize melatonin to AFMK. We conclude that neutrophils are one of the main targets for melatonin and that at least part of the effects described for melatonin on immune cells may be due to its oxidation product, AFMK. We also consider that the oxidation of melatonin may be an important event in the cross-talking between neutrophils and monocytes.

The murine hair follicle is a melatonin target

The pineal hormone, melatonin exerts many functional effects on mammalian skin (e.g. melanogenesis inhibition, melanocyte growth inhibition, and regulation of seasonal pelage hair growth). However, its cutaneous expression, regulation, and functional role are still obscure. The aim of this study was to check whether murine hair follicles are indeed direct, peripheral melatonin targets which express melatonin membrane receptors (MT1 and MT2) and orphan nuclear receptor α (RORα) which interact with melatonin. Immunohistochemistry revealed that murine hair follicle keratinocytes show both MT1‐like immunoreactivity (IR) and ROR‐like IR, both of which changed substantially in a hair cycle‐dependent manner. Both semiquantitive RT‐PCR for MT1 and MT2, and quantitive real‐time PCR for MT1, MT2, and ROR on murine skin cDNA revealed that all three genes are transcribed in normal mouse skin in hair cycle‐dependent manner. Functionally, melatonin significantly inhibited the constitutional level of epidermal and hair follicle keratinocyte apoptosis in short‐term mouse skin organ culture. In conclusion, we here provide evidence that normal murine hair follicles are prominent direct target for melatonin bioregulation which express MT1, MT2, and ROR, at least some of which are functionally active in situ. These receptors are regulated in a hair cycle‐dependent manner, suggesting a role of melatonin in hair cycle control.

Activity of the hippocampal somatostatinergic system following daily administration of melatonin

If melatonin or its analogs are to be used therapeutically in humans, their chronic effects on responsiveness of melatonin target cells need to be assessed. We have previously demonstrated that acute melatonin treatment regulates the somatostatinergic system in the rat hippocampus. In the present study, we have investigated the effects of subchronic and chronic daily treatment with melatonin on the somatostatinergic system in the rat hippocampus. Male Wistar rats (200–250 g) were injected with melatonin (25 μg/kg body weight, subcutaneously) daily for 4, 7 or 14 days and sacrificed 24 h after the last injection. Melatonin administration for 4 days induced a decrease in the hippocampal somatostatin (SRIF)-like immunoreactivity content as well as a decrease in the number of SRIF receptors and an increase in their apparent affinity. The decreased number of SRIF receptors in the melatonin (4 days)-treated rats was associated with a decreased capacity of SRIF to inhibit both basal and forskolin-stimulated adenylyl cyclase activity. These melatonin-induced effects reversed to control values after 7 or 14 days of treatment. Hippocampal membranes from control and melatonin-treated rats showed similar Gi and Gs activities. Melatonin treatment altered neither the functional Gi activity nor the Giα1 or Giα2 levels at any of the time periods studied. The present results suggest that chronic exposure to melatonin results in a tolerance of the hippocampus to this hormone.

The premammillary hypothalamic area of the ewe: anatomical characterization of a melatonin target area mediating seasonal reproduction.

Recent evidence suggests that the ovine premammillary hypothalamic area (PMH) is an important target for the pineal hormone, melatonin, and its role in seasonal reproduction. In rodents, the PMH is a complex region consisting of several cell groups with differing neurochemical content and anatomical connections. Therefore, to obtain a better understanding of the potential neural targets for melatonin in this area of the sheep brain, we have undertaken a detailed anatomical characterization of the PMH, including its nuclear divisions and the location of neuropeptide/neurotransmitter cells within them. By combining immunocytochemistry for NeuN, a neuronal marker, with Nissl staining in anestrous, ovariectomized, estradiol-treated ewes, we identified three nuclei within the PMH: a caudal continuation of the hypothalamic arcuate nucleus (cARC), the ventral division of the premammillary nucleus (PMv), and the ventral tuberomammillary nucleus (TMv). The cARC contained neurons that were immunoreactive for tyrosine hydroxylase, dynorphin, estrogen receptor alpha, cocaine- and amphetamine-regulated transcript peptide (CART), and nitric oxide synthase (NOS). The PMv was also characterized by the presence of cells that contained NOS and CART, although the size of these cells was larger than that of their corresponding phenotype in the cARC. By contrast, in the TMv, of the markers examined in the present study, only fibers immunoreactive for orexin were seen. Thus, the ovine PMH is a heterogeneous region comprised of three subdivisions, each with distinct morphological and neurochemical characteristics. This anatomical map of the PMH provides a basis for future studies to determine the functional contribution of each component to the influence of melatonin on seasonal reproduction.

Evidence for an endogenous per1- and ICER-independent seasonal timer in the hamster pituitary gland.

Most mammals use changing annual day-length cycles to regulate pineal melatonin secretion and thereby drive many physiological rhythms including reproduction, metabolism, immune function, and pelage. Prolonged exposure to short winter day lengths results in refractoriness, a spontaneous reversion to long-day physiological status. Despite its critical role in the timing of seasonal rhythms, refractoriness remains poorly understood. The aim of this study was therefore to describe cellular and molecular mechanisms driving the seasonal secretion of a key hormone, prolactin, in refractory Syrian hamsters. We used recently developed single cell hybridization and reporter assays to show that this process is initiated by timed reactivation of endocrine signaling from the pars tuberalis (PT) region of the pituitary gland, a well-defined melatonin target site, causing renewed activation of prolactin gene expression. This timed signaling is independent of per1 clock gene expression in the suprachiasmatic nuclei and PT and of melatonin secretion, which continue to track day length. Within the PT, there is also a continued short day-like profile of ICER expression, suggesting that the change in hormone secretion is independent of cAMP signaling. Our data thus identify the PT as a key anatomical structure involved in endogenous seasonal timing mechanisms, which breaks from prevailing day length-induced gene expression.

The human myometrium as a target for melatonin.

The circadian timing of spontaneous human deliveries results in births occurring statistically more often during the nocturnal phase of the 24-h cycle. The neuroendocrine mechanisms underlying this physiological phenomenon are not understood. In an effort to test the hypothesis that melatonin may serve as an endocrine signal for coordinating myometrial events in the human, we determined the mRNA expression of both MT1 and MT2 melatonin receptor isoforms in pregnant as well as nonpregnant myometrial biopsies by means of RT-PCR and in situ hybridization histochemistry. Additionally, we could demonstrate specific, high affinity iodomelatonin binding to myometrial tissues of both pregnant and nonpregnant women. Primary cultures of myocytes responded differentially from melatonin in terms of cAMP signaling depending on the reproductive state. These results imply that melatonin may have the potential to modulate myometrial function in the human, a finding that could open up new possibilities for the development of novel therapeutic agents.

Seasonality of food intake in ruminants: recent developments in understanding

Domestic ruminants are used to exploit many vegetation resources that would otherwise be unproductive. For maximal effectiveness, there is a need to understand underlying mechanisms controlling animal performance, including seasonal variations in appetite and food intake. Potentially useful experimental approaches, recent findings and aspects for future study are discussed. Seasonal variation in intake is expressed through changes in the pattern of meals (duration, frequency, inter-meal interval and ingestion rate). These changes are signalled through alterations in both structure and function of the gastrointestinal tract and physiological signals. Studies suggest that multiple, interactive signals are involved, including hormones such as cholecystokinin, insulin, leptin and triiodothyronine. However, baseline concentrations in the peripheral circulation are not appropriate measurements of some of these hormones since there can be seasonal differences in postprandial profiles or changes in rate of dilution in the bloodstream or in the rate of degradation in the liver. Interactions between these circulating signals, liver function and neural signals to the brain need clarification. Systemic nutritional signals also act directly in the brain where they are integrated with seasonal photoperiod (melatonin) signalling within the hypothalamus. Melatonin target sites critical to appetite regulation have still to be identified, but leptin receptors and downstream neuropeptides have been localised within the ovine hypothalamus. These orexigenic and anorexigenic 'compensatory' pathways are sensitive to imposed changes in nutritional status but, with the exception perhaps of cocaine- and amphetamine-regulated transcript, do not appear to drive seasonal 'anticipatory' changes in intake. Mechanisms underlying seasonal changes in hypothalamic sensitivity to nutritional feedback clearly deserve further study.

Melatonin Receptor mRNA and Protein Expression in Xenopus laevis Nonpigmented Ciliary Epithelial Cells

Melatonin is an output signal of the circadian clock, and may regulate diurnal rhythms in ocular tissues. A role for melatonin has been suggested in the circadian changes in intraocular pressure (IOP). Changes in IOP may be due partially to changes in the rate of aqueous humor secretion, which is produced by the nonpigmented epithelium of the ciliary body. To examine the mechanism by which melatonin may influence ciliary epithelium function and perhaps the IOP diurnal rhythm, immunocytochemistry with an antibody directed against the Mel1c melatonin receptor subtype was performed on sections of Xenopus eyes. Melatonin receptor immunoreactivity was observed in the basolateral regions of the nonpigmented epithelial cells of the ciliary body. Receptor immunoreactivity was also observed in cells of the retina, as has been previously reported. Specific immunoreactivity was not observed in the epithelium of the iris or pigmented ciliary epithelium. In situ hybridization of the Xenopus eye revealed expression of Mel1c but not Mel1b receptor mRNA in the nonpigmented ciliary epithelium. These results provide evidence that the nonpigmented epithelia of the ciliary body are direct targets for melatonin, and supports previous work that melatonin may influence the rate of aqueous humor secretion by ciliary epithelium, and perhaps the circadian rhythm of IOP.

Mammalian photoperiodic system: formal properties and neuroendocrine mechanisms of photoperiodic time measurement.

Photoperiodism is a process whereby organisms are able to use both absolute measures of day length and the direction of day length change as a basis for regulating seasonal changes in physiology and behavior. The use of day length cues allows organisms to essentially track time-of-year and to "anticipate" relatively predictable annual variations in important environmental parameters. Thus, adaptive types of seasonal biological changes can be molded through evolution to fit annual environmental cycles. Studies of the formal properties of photoperiodic mechanisms have revealed that most organisms use circadian oscillators to measure day length. Two types of paradigms, designated as the external and internal coincidence models, have been proposed to account for photoperiodic time measurement by a circadian mechanism. Both models postulate that the timing of light exposure, rather than the total amount of light, is critical to the organism's perception of day length. In mammals, a circadian oscillator(s) in the suprachiasmatic nucleus of the hypothalamus receives photic stimuli via the retinohypothalamic tract. The circadian system regulates the rhythmic secretion of the pineal hormone, melatonin. Melatonin is secreted at night, and the duration of secretion varies in inverse relation to day length; thus, photoperiod information is "encoded" in the melatonin signal. The melatonin signal is presumably "decoded" in melatonin target tissues that are involved in the regulation of a variety of seasonal responses. Variations in photoperiodic response are seen not only between species but also between breeding populations within a species and between individuals within single breeding populations. Sometimes these variations appear to be the result of differences in responsiveness to melatonin; in other cases, variations in photoperiod responsiveness may depend on differences in patterns of melatonin secretion related to circadian variation. Sites of action for melatonin in mammals are not yet well characterized, but potential targets of particular interest include the pars tuberalis of the pituitary gland and the suprachiasmatic nuclei. Both these sites exhibit uptake of radiolabeled melatonin in various species, and there is some evidence for direct action of melatonin at these sites. However, it appears that there are species differences with respect to the importance and specific functions of various melatonin target sites.

A role for tyrosine phosphorylation in the regulation and sensitization of adenylate cyclase by melatonin.

Mimicking short photoperiod melatonin signals (16 h exposure) on primary cell cultures of melatonin target cells of the ovine pars tuberalis (PT) results in an enhanced cAMP response to forskolin stimulation relative to untreated cells, a phenomenon termed sensitization. The sensitized response of PT cells may be an important aspect of the interpretation of the melatonin signal to initiate appropriate seasonal physiological responses. The aim of this study is to add to our understanding of the molecular mechanisms involved in the sensitization of PT cells by melatonin. We demonstrate that sensitization of PT cells by melatonin is mediated via a G(i)-coupled melatonin receptor. The sensitized cAMP response is not only obtained with the pharmacological tool forskolin, but also with cholera toxin, an activator of G(salpha). Changes in the level of G(salpha) or G(ialpha) G-protein subunits are ruled out as part of the sensitization mechanism. However, changes in tyrosine phosphorylation may be involved as tyrosine kinase inhibitors sensitize ovine PT cells and tyrosine phosphatase inhibitors significantly blunt adenylate cyclase activity, including the sensitized response to melatonin. The adenylate cyclase isoforms mediating the sensitized response may be broad as 7 of the 9 isoforms of adenylate cyclase are expressed in the PT.

A role for tyrosine phosphorylation in the regulation and sensitization of adenylate cyclase by melatonin

Mimicking short photoperiod melatonin signals (16 h exposure) on primary cell cultures of melatonin target cells of the ovine pars tuberalis (PT) results in an enhanced cAMP response to forskolin stimulation relative to untreated cells, a phenomenon termed sensitization. The sensitized response of PT cells may be an important aspect of the interpretation of the melatonin signal to initiate appropriate seasonal physiological responses. The aim of this study is to add to our understanding of the molecular mechanisms involved in the sensitization of PT cells by melatonin. We demonstrate that sensitization of PT cells by melatonin is mediated via a Gj-coupled melatonin receptor. The sensitized cAMP response is not only obtained with the pharmacological tool forskolin, but also with cholera toxin, an activator of Gsα. Changes in the level of GsQ, or Giα G-protein subunits are ruled out as part of the sensitization mechanism. However, changes in tyrosine phosphorylation may be involved as tyrosine kinase inhibitors sensitize ovine PT cells and tyrosine phosphatase inhibitors significantly blunt adenylate cyclase activity, including the sensitized response to melatonin. The adenylate cyclase isoforms mediating the sensitized response may be broad as 7 of the 9 isoforms of adenylate cyclase are expressed in the PT.–Barrett, P., Choi, W.-S., Morris, M., Morgan, P. A role for tyrosine phosphorylation in the regulation and sensitization of adenylate cyclase by melatonin. FASEB J. 14, 1619–1628 (2000)

Dual Signaling of Human Mel1a Melatonin Receptors via Gi2, Gi3, and Gq/11 Proteins

Mel 1a melatonin receptors belong to the super-family of guanine nucleotide-binding regulatory protein (G protein)-coupled receptors. So far, interest in Mel 1a receptor signaling has focused mainly on the modulation of the adenylyl cyclase pathway via pertussis toxin (PTX)-sensitive G proteins. To further investigate signaling of the human Mel 1a receptor, we have developed an antibody directed against the C terminus of this receptor. This antibody detected the Mel 1a receptor as a protein with an apparent molecular mass of approximately 60 kDa in immunoblots after separation by SDS-PAGE. It also specifically precipitated the 2-[125I]iodomelatonin (125I-Mel)-labeled receptor from Mel 1a-transfected HEK 293 cells. Coprecipitation experiments showed that G(i2), G(i3), and G(q/11) proteins couple to the Mel 1a receptor in an agonist-dependent and guanine nucleotide-sensitive manner. Coupling was selective since other G proteins present in HEK 293 cells, (G(i1), G(o), G(s), G(z), and G12) were not detected in receptor complexes. Coupling of the Mel 1a receptor to G(i) and G(q) was confirmed by inhibition of high-affinity 125I-Mel binding to receptors with subtype-selective G protein alpha-subunit antibodies. G(i2) and/or G(i3) mediated adenylyl cyclase inhibition while G(q/11) induced a transient elevation in cytosolic calcium concentrations in HEK 293 cells stably expressing Mel 1a receptors. Melatonin-induced cytosolic calcium mobilization via PTX-insensitive G proteins was confirmed in primary cultures of ovine pars tuberalis cells endogenously expressing Mel 1a receptors. In conclusion, we report the development of the first antibody recognizing the cloned human Mel 1a melatonin receptor protein. We show that Mel 1a receptors functionally couple to both PTX-sensitive and PTX-insensitive G proteins. The previously unknown signaling of Mel 1a receptors through G(q/11) widens the spectrum of potential targets for melatonin.

Expression of mt 1 melatonin receptor in rat retina: evidence for multiple cell targets for melatonin

Melatonin is synthesized in the retina at night and acts as a local modulator within this tissue by mediating the effects of darkness. We investigated the expression and localization of the mt 1 (Mel 1a) melatonin receptor in rat retina in order to disclose the cellular and molecular bases of melatonin's action in the mammalian retina. Western blotting of the mt 1 receptor in rat retina exhibited a single immunoreactive band of approximately 37,000 mol. wt, which corresponds to the predicted molecular size of the receptor. The mt 1 receptor was immunocytochemically localized to both the inner and outer plexiform layers. During postnatal development, retina from two-week-old rats showed the highest mt 1 immunoreactivity; the outer plexiform layer and horizontal cell bodies were strongly immunolabeled, with weaker labeling in the inner plexiform layer. Expression of mt 1 receptor messenger RNA in the rat retina was demonstrated by reverse transcription–polymerase chain reaction and in situ hybridization. mt 1 receptor transcripts were localized to ganglion cells, amacrine cells and horizontal cells. These results suggest that melatonin influences retinal physiology by acting on multiple retinal cell types, including ganglion, amacrine and horizontal cells, via the mt 1 receptor expressed in their processes.

Melatonin directly suppresses steroid production by preovulatory follicles in the cyclic hamster.

The purpose of these studies was to investigate the effects of melatonin on the production of steroids (progesterone, testosterone, and estradiol) and cAMP by preovulatory follicles and to examine changes in melatonin concentrations in the ovary during the estrous cycle. Adult cyclic hamsters were used in this study. Melatonin concentrations in the ovary, pineal gland, and serum were measured at mid-light and mid-dark during the estrous cycle. Effects of melatonin on steroidogenesis by preovulatory follicles, thecae, and granulosa cells were examined, and its effect on cAMP production by preovulatory follicles was also investigated. Melatonin (0.1-10 ng/ml) had no effect on steroid production in the absence of hCG, but melatonin decreased progesterone and estradiol production by preovulatory follicles in a dose-dependent and time-dependent manner in the presence of hCG (100 mIU/ml). The target of melatonin was thecae but not granulosa cells, and melatonin significantly reduced cAMP production by preovulatory follicles. Melatonin concentrations in the ovary showed a similar phasic variation with high levels during mid-dark and low during mid-light, as in the pineal gland and serum. These results show that the ovarian melatonin levels also exhibit a circadian rhythm and suggest that the high melatonin milieu in the ovary may induce gonadal regression in the cyclic hamster.

Evidence that melatonin acts in the premammillary hypothalamic area to control reproduction in the ewe: presence of binding sites and stimulation of luteinizing hormone secretion by in situ microimplant delivery.

Melatonin transduces the effect of day length on LH secretion by acting on the hypothalamus. However, the precise hypothalamic site is unknown. Two studies were undertaken to clarify where melatonin acts in the hypothalamus. Using autoradiographic methods, the hypothalami of 5 ewes were screened to determine whether specific regional densities in melatonin binding existed. A higher density of binding was observed in the premammillary area of the hypothalamus (PMH) (3- to 5-fold higher than the rest of the hypothalamus). This binding area is delimited rostrally by the infundibular recess, caudally by the mammillary bodies, dorsally by the fornix, and ventrally by the base of the brain; and it encompasses the premammillary and tuberomammillary nuclei. To test the functional importance of the identified area, 3 groups of animals received bilateral melatonin microimplants: 1) in the PMH (n = 11); 2) in the anterior/mediobasal hypothalamus (AH/MBH; n = 8); and 3) sham-operated animals received empty microimplants in the PMH (SHAM; n = 6). All ewes were ovariectomized and treated s.c. with a 20-mm SILASTIC brand capsule of estradiol and exposed to long days (16-h light, 8-h dark). At the end of the 80-day experiment, no animal of the SHAM group and only 2 of the 8 ewes of the AH/MBH group displayed a stimulation of LH secretion. In contrast, melatonin implanted in the PMH stimulated LH secretion in 10 of the 11 ewes on day 44.5 +/- 5.3 (mean +/- SEM). ANOVA revealed that the changes in LH secretion were not different between the SHAM and the AH/MBH groups but the PMH group differed from the other 2 groups (P < 0.0001). This study suggests that the PMH is an important target for melatonin to regulate reproductive activity.

The pineal organ, its hormone melatonin, and the photoneuroendocrine system.

The vertebrate pineal organ rhythmically synthesizes and secretes melatonin during nighttime and forms an essential component of the photoneuroendocrine system which allows humans and animals to measure and keep the time. Regulation of the melatonin biosynthesis depends on signals from photoreceptors perceiving and transmitting environmental light stimuli and endogenous oscillators generating a circadian rhythm which is independent from any environmental time cue (zeitgeber). In nonmammalian species the photoreceptors responsible for regulating melatonin biosynthesis reside within the pineal organ itself. In several nonmammalian species (e.g., lamprey, zebra fish, house sparrow, chicken) the pineal organ is also capable of generating circadian rhythms and thus serves all key functions of the photoneuroendocrine system: photoreception, endogenous rhythm generation, and production of neurohormones. These may even be accomplished by a single "photoneuroendocrine" cell. In mammals the pineal organ has lost both the direct light sensitivity and the capacity of generating circadian rhythms, and melatonin biosynthesis is regulated by retinal photoreceptors and a circadian oscillator located in the suprachiasmatic nucleus of the hypothalamus. Due to this spatial separation the photoneuroendocrine system of mammals comprises neuronal and neuroendocrine pathways which interconnect its components. The neuronal pathways involve circuits of both the central and the peripheral nervous systems, and as an important final link noradrenergic sympathetic nerve fibers. The suprachiasmatic nucleus appears as a major target of melatonin in mammals. The pineal hormone may thus be involved in a feedback loop of the mammalian photoneuroendocrine system. The present comparative contribution considers, after a short survey of classical findings on the phylogenetic development and the gross anatomy of the pineal complex, cytoevolutionary and cell biological aspects of the various types of pinealocytes as well as the afferent and efferent innervation of the pineal organ (pinealofugal and pinealopetal neuronal pathways). Moreover, emphasis is placed on receptor mechanisms, second messenger systems (Ca2+ and cyclic AMP), transcription factors (e.g, CREB and ICER), and their roles for regulation of melatonin biosynthesis. Finally, the action, targets, and receptors of melatonin are dealt with. The synoptic approach of this contribution, which combines anatomical and ultrastructural findings with cell and molecular biological results, confirms the functional significance of the melatonin-synthesizing pineal organ as an important component of the photoneuroendocrine system and stresses the importance of this organ as a model to study signal transduction mechanisms both in photoreceptors and in neuroendocrine cells.

Endocrinology meets immunology: T lymphocytes as novel targets for melatonin.

Endocrinology meets immunology: T lymphocytes as novel targets for melatonin.

High-affinity binding of melatonin by human circulating T lymphocytes (CD4+).

This paper shows the presence of high-affinity binding sites for melatonin in human circulating T lymphocytes, but not in B lymphocytes. The binding of melatonin to T cells was dependent on time, stable, reversible, saturable, specific, and inversely correlated to the production of melatonin, expressed as the nocturnal 12 h production of its urinary metabolite 6-sulfatoxymelatonin. The affinity of these binding sites (Kd = 0.27 nM) suggests that they may recognize the physiological concentrations of melatonin in serum. Moreover, among the lymphocyte subpopulations studied, binding of melatonin was mostly found in CD4+ cells rather than in CD8+ cells. Results suggest that CD4+ cells may be the target of melatonin among the human circulating lymphocytes.

Distribution and characterization of the melatonin receptors in the hypothalamus and pituitary gland of three domestic ungulates.

With some exceptions, in most of the mammals the pituitary pars tuberalis and the hypothalamic suprachiasmatic nuclei are reportedly the main targets for the pineal hormone melatonin. However, it is not known if the conspicuous diversity in the distribution pattern of melatonin binding sites in these areas depicts differences in reproductive behavior observed in the seasonally breeding species in the temperate zones. We explored the distribution and the characteristics of melatonin binding sites in the hypothalamus and pituitary of three species (bovine, horse, and donkey) different in terms of seasonal reproductive competence. The topographical localization, investigated by in vitro autoradiography, revealed 2-[125I]iodomelatonin binding sites only in the pituitary gland in all three species, primarily in the pars tuberalis (PT), but also in the pars distalis (PD) and pars intermedia (PI). Kinetic, inhibition, and saturation studies, performed by means of in vitro binding, revealed presence of a single class high affinity binding sites. The Kd values, melatonin, and 2-iodomelatonin Ki values were in the low picomolar range. Coincubation with GTP gamma S inhibited 2-[125I]iodomelatonin binding, demonstrating that these putative receptors are linked to a G protein in their signal-transduction pathway. The hypothalamus was devoid of specific binding. In conclusion, the results suggest that in these species, the hypophysis may be a principal target for the melatonin action on the reproductive system.

T-helper-2 lymphocytes as a peripheral target of melatonin.

In the past several years we demonstrated that the pineal neurohormone melatonin has immunoenhancing properties and can counteract the immunodepression that may follow acute stress, drug treatment, and viral diseases or aging. Several laboratories have subsequently confirmed and extended our findings. It soon appeared evident that T-derived cytokines constitute the main mediators of the immunological effect of melatonin. We have recently found a high affinity (Kd: 346 +/- 24 pM) binding site for 125I-melatonin on T-helper-type 2 lymphocytes in the bone marrow. Activation of this putative melatonin receptor, with both physiological and pharmacological concentrations of melatonin, resulted in an enhanced production of interleukin-4 (IL4), which in turn acted on bone marrow stromal cells and induced the release of hematopoietic growth factors. This melatonin-cytokine cascade showed the remarkable capacity of rescuing hematopoietic functions in mice treated with cancer chemotherapeutic compounds without interfering with the anticancer action of these agents. The very low concentration (0.1 nM) at which melatonin is active may well reflect a physiological function of endogenous melatonin. The pineal gland has been, in fact, reported to signal the blood forming system. The evidence of IL4 involvement is relevant to our understanding of many melatonin effects and may be part of a pineal-immune axis involving also Th1 cytokines. The ability of rescuing hematopoiesis against the toxic action of cancer chemotherapeutic compounds and the presence of high-affinity IL4 receptors on human tumors provide a further promising rationale for the clinical use of melatonin.