Peripheral Melatonin Research Articles

Peripheral melatonin modulates seasonal immunity and reproduction of Indian tropical male bird Perdicula asiatica

Seasonal changes in pineal function are well coordinated with seasonal reproductive activity of tropical birds. Further, immunomodulatory property of melatonin is well documented in seasonally breeding animals. Present study elucidates the interaction of peripheral melatonin with seasonal pattern of immunity and reproduction in Indian tropical male bird Perdicula asiatica. Significant seasonal changes were noted in pineal, testicular and immune function(s) of this avian species. Maximum pineal activity along with high immune status was noted during winter month while maximum testicular activity with low immune status was noted in summer. During summer month's long photoperiod suppressed pineal activity and high circulating testosterone suppressed immune parameters, while in winter short photoperiod elevated pineal activity and high circulating melatonin maintained high immune status and suppressed gonadal activity. Therefore, seasonal levels of melatonin act like a major temporal synchronizer to maintain not only the seasonal reproduction but also immune adaptability of this avian species.

Annual reproductive synchronization in ovary and pineal gland function of female short-nosed fruit bat, Cynopterus sphinx

We studied the annual correlation of ovarian activity and pineal gland in relation with seasonal variation and gestation of a tropical zone short-nosed fruit bat Cynopterus sphinx. Female bats showed bimodal polyestry (February/March and September/October) in their reproductive cycle. Plasma estradiol concentration ran parallel with ovarian activity and had an inverse relation with pineal mass and peripheral melatonin concentration. Due to the delayed embryonic development in the uterus (October–March) of female bats, interestingly, the uterine activity did not show a parallel relation with ovarian activity and estradiol level. Further, compared with normal non-pregnant females, melatonin level was high during gestation and delayed embryonic development phase. This suggests that the reproductive synchrony and annual variation in ovarian activity of this nocturnal flying mammal differ from other common tropical mammals. The delayed embryonic development in bats might be an adaptive strategy for the unfavorable conditions of the seasons and might be regulated by high peripheral estradiol and melatonin concentration.

Melatonin and differential effect of l-thyroxine on immune system of Indian tropical bird Perdicula asiatica

Interaction of thyroxine and melatonin on immune status was noted in vivo and in vitro when peripheral melatonin was high and thyroxine low in plasma of male Perdicula asiatica during reproductively inactive phase. During this phase exogenous thyroxine (4 μg/100 g. Bwt./day) and melatonin (25 μg/100 g. Bwt./day) increased immune parameters (spleen weight, total leukocyte count, lymphocyte count, percent stimulation ratio) and increased splenocyte density in spleen. In vitro l-thyroxine (10 −6 M/ml) supplementation decreased the splenocyte proliferation which was reversed by melatonin (500 pg/ml) supplementation. In vivo l-thyroxine showed immunoenhancing effect while in vitro it decreased the splenocyte proliferation presenting a differential effect. In the absence of internal physiological conditions of the birds, T 4 showed a negative effect on splenocytes proliferation in vitro when treated alone. However, melatonin maintained its lymphoproliferative effect under both conditions. Thus, avian splenocyte exposed to different hormonal conditions in vitro might have produced different signal peptides other than in vivo, thereby making the result different.

A role of melatonin in neuroectodermal‐mesodermal interactions: the hair follicle synthesizes melatonin and expresses functional melatonin receptors

Since mammalian skin expresses the enzymatic apparatus for melatonin synthesis, it may be an extrapineal site of melatonin synthesis. However, evidence is still lacking that this is really the case in situ. Here, we demonstrate melatonin-like immunoreactivity (IR) in the outer root sheath (ORS) of mouse and human hair follicles (HFs), which corresponds to melatonin, as shown by radioimmunoassay and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The melatonin concentration in organ-cultured mouse skin, mouse vibrissae follicles, and human scalp HFs far exceeds the respective melatonin serum level and is significantly increased ex vivo by stimulation with norepinephrine (NE), the key stimulus for pineal melatonin synthesis. By real-time PCR, transcripts for the melatonin membrane receptor MT2 and for the nuclear mediator of melatonin signaling, retinoid orphan receptor alpha (ROR)alpha, are detectable in murine back skin. Transcript levels for these receptors fluctuate in a hair cycle-dependent manner, and are maximal during apoptosis-driven HF regression (catagen). Melatonin may play a role in hair cycle regulation, since its receptors (MT2 and RORalpha) are expressed in murine skin in a hair cycle-dependent manner, and because it inhibits keratinocyte apoptosis and down-regulates ERalpha expression. Therefore, the HF is both, a prominent extrapineal melatonin source, and an important peripheral melatonin target tissue. Regulated intrafollicular melatonin synthesis and signaling may play a previously unrecognized role in the endogenous controls of hair growth, for example, by modulating keratinocyte apoptosis during catagen and by desensitizing the HF to estrogen signaling. As a prototypic neuroectodermal-mesodermal interaction model, the HF can be exploited for dissecting the obscure role of melatonin in such interactions in peripheral tissues.

Importance of the pineal gland, endogenous prostaglandins and sensory nerves in the gastroprotective actions of central and peripheral melatonin against stress‐induced damage

Melatonin attenuates acute gastric lesions induced by topical strong irritants because of scavenging of free radicals, but its role in the pathogenesis of stress-induced gastric lesions has been sparingly investigated. In this study we compared the effects of intragastric (i.g.) or intracerebroventricular (i.c.v.) administration of melatonin and its precursor, L-tryptophan, with or without concurrent treatment with luzindole, a selective antagonist of melatonin MT2 receptors, on gastric lesions induced by water immersion and restraint stress (WRS). The involvement of pineal gland, endogenous prostaglandins (PG) and sensory nerves in gastroprotective action of melatonin and L-tryptophan against WRS was studied in intact or pinealectomized rats or those treated with indomethacin or rofecoxib to suppress cyclooxygenase (COX)-1 and COX-2, respectively, and with capsaicin to induce functional ablation of the sensory nerves. In addition, the influence of i.c.v. and i.g. melatonin on gastric secretion was tested in a separate group of rats equipped with gastric fistulas. At 3.5 hr after the end of WRS, the number of gastric lesions was counted, the gastric blood flow (GBF) was determined by H2-gas clearance technique and plasma melatonin and gastrin levels were measured by specific radioimmunoassay (RIA). Biopsy mucosal samples were taken for determination of expression of mRNA for COX-1 and COX-2 by reverse transcriptase-polymerase chain reaction (RT-PCR) and of the mucosal generation of prostaglandin E2 (PGE2) by RIA. Melatonin applied i.g. (1.25-10 mg/kg) or i.c.v. (1.25-10 microg/kg) dose-dependently inhibited gastric acid secretion and significantly attenuated the WRS-induced gastric damage. This protective effect of melatonin was accompanied by a significant rise in the GBF and plasma melatonin and gastrin levels and in mucosal generation of PGE2. Pinealectomy, which suppressed plasma melatonin levels, aggravated the gastric lesions induced by WRS and these effects were counteracted by i.g. or i.c.v. application of melatonin. Luzindole abolished completely the gastroprotective effects of melatonin and L-tryptophan and attenuated significantly the rise in GBF evoked by the indoleamine and its precursor. Indomethacin and rofecoxib, which diminished PGE2 biosynthesis by c. 90 and 75% or capsaicin denervation, attenuated significantly melatonin- and L-tryptophan-induced protection and the rise in the GBF. Both the protection and the hyperemia were restored by addition of exogenous CGRP to capsaicin-denervated animals. COX-1 mRNA was detected by RT-PCR in the intact and melatonin-treated gastric mucosa, while COX-2 mRNA, which was undetectable in the intact gastric mucosa, appeared in WRS-exposed mucosa, especially in the melatonin-treated animals and this was accompanied by increased generation of PGE2 in gastric mucosa. Pinealectomy downregulated COX-2 mRNA and this effect was reversed by supplementation of pinealectomized animals with melatonin. We conclude that, (a) exogenous melatonin and its precursor, L-tryptophan, attenuates WRS-induced gastric lesions via interaction with MT2 receptors, (b) this protective action of melatonin is because of an enhancement of gastric microcirculation, probably mediated by PGE2 derived from COX-2 overexpression and activity, the activation of brain-gut axis involving CGRP released from sensory nerves, and the release of gastrin and (c) the pineal plays an important role in the limitation of WRS-induced gastric lesions via releasing melatonin, which exerts gastroprotective and hyperemic activities against stress ulcerogenesis.

Expression and functional relevance of melatonin receptors in hair follicle biology

The prototypic pineal hormone, melatonin reputedly exerts many functional effects on mammalian skin and/or its isolated cell populations in culture (e.g., melanogenesis inhibition, melanocyte growth inhibition, regulation of seasonal changes in the pelage), and is recognized as a potent free radical scavenger. In mammals, two types of high‐affinity membrane melatonin receptors, MT1 and MT2 have been identified, which inhibit adenylate cyclase activity to decrease the intracellular level of cAMP. Low‐affinity membrane receptor MT3/QR2 have also been identified, though the mechanism has not been cleared yet. Melatonin is also a natural ligand of nuclear transcription factor ROR(α and β), which is suggested to regulate cell cycle negatively via target gene such as p21WAF/CIP1. Due to its lipophilic structure, melatonin also enters through both the plasma and nuclear membrane, and acts as a potent free radical scavenger to protect macromolecules, in particular DNA. Melatonin demonstrates differential – and often still confusing seemingly contradictory– effects on cell activity in many different systems, which may be explained by this multitude of signaling pathways that are modulated by melatonin bioactivity. Recently, cultured epidermal and follicular melanocytes, keratinocytes, and fibroblasts, have also been found to display the enzymatic activity of arylalkylamine N‐acetyltransferase and hydroxyindole ‐O‐ methyltransferase for melatonin synthesis. However, little is known about the cutaneous expression and regulation of melatonin and its receptors in situ, and the functional role of melatonin in normal skin and hair follicle biology is still obscure. In order to study whether murine hair follicles in situ are indeed direct peripheral melatonin targets, the follicular expression of MT1, MT2 and/or RORα are investigated. Immunohistochemistry revealed that C57BL/6 mouse hair follicle keratinocytes in situ show prominent MT1‐like immunoreactivity (IR), which changed substantially in a hair cycle‐dependent manner. RORα‐like IR was also detected in murine hair follicles, and also displayed hair cycle dependence. RT‐PCR of MT1 and MT2, and real time PCR for MT1, MT2 and RORα on C57BL/6 mice skin cDNA revealed that all three genes are transcribed in normal mouse skin, and demonstrated that their expression/transcription is hair cycle‐dependent. In conclusion, normal murine hair follicles are indeed a prominent, direct target for melatonin bioregulation, through MT1, MT2 and RORα melatonin receptors and that at least some of these regulators are functionally active in situ. The observed hair cycle dependence of melatonin receptor expression suggests a role of melatonin in hair cycle control.

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.

Melatonin and the circadian rhythms of feeding and perch-hopping in the European starling,Sturnus vulgaris

The role of melatonin plasma titers in the control of free-running circadian rhythms was investigated in European starlings,Sturnus vulgaris, held in continuous dim light. Simultaneous recordings of plasma melatonin, perch-hopping and feeding activity revealed synchronous circadian variations in all three functions with high melatonin titers during resting and low titers during activity periods. Implanting birds with melatonin silastic capsules resulted in a 100-fold increase in plasma concentrations and an abolition of the endogenous melatonin rhythm. Feeding rhythms persisted during this treatment, although the activity time (α) was lengthened and the circadian period (τ) shortened. Similar changes were found in perchhopping activity, but in some birds, this activity was more or less suppressed. These data demonstrate that although melatonin plasma titers freerun synchronously with other circadian parameters, fluctuations in peripheral melatonin are not essential for the expression of circadian activity.

A rise in peripheral melatonin levels induces ovarian activity in anestrous sheep.

Melatonin in Silastic capsules was implanted subcutaneously to examine the effect on reproductive activities of anestrous ewes under natural photoperiods. The constant supply of melatonin from the capsules increased plasma melatonin levels in a dose dependent manner, and high concentrations exceeding the endogenous night peaks were maintained throughout the day in the ewes carrying two capsules. On May 8 (Day 0) non-lactating ewes (n=23) were divided into 3 groups, and were either implanted with one (MEL 1, n=7) or two (MEL 2, n=6) capsules, or left untreated (control, n=10). Plasma progesterone concentrations in the MEL 2 group rose and became significantly higher than those in the control group from Day 56 onwards. This suggests a stimulatory effect of melatonin on quiescent ovarian function. In the MEL 1 group, however, no apparent effect was observed, probably due to an insufficiency in basal melatonin ele-vation. These results suggest that the continuous presence of high melatonin levels in the circulation nullified the anti-gonadal effect of long days, presumably by blunting the endogenous rhythm of melatonin levels responsible for conveying information of the environmental photoperiod.

Oral administration and distribution of melatonin in human serum, saliva and urine

In order to evaluate for future physiological and pharmacological studies the extent to which orally administered melatonin is found in human serum and saliva and excreted into urine we measured serum, saliva and urine concentrations of melatonin by radioimmunoassau after oral administration of 100 mg melatonin. Elevated melatonin concentrations were observed with peak values of 435 nmol/l in serum and 241 nmol/l in saliva at 60 min. Elimination was monophasic following first-order kinetics. The half-lives for serum and saliva melatonin were 41 and 38 min, respectively. The results suggest that melatonin is passively secreted into saliva which reflects closely the changes in serum melatonin. Saliva sampling is thus useful in studies on peripheral melatonin both in physiological and experimental conditions. Urinary excretion of melatonin was 0.01 % of the amount of melatonin ingested. In high-performance liquid chromatography urine extracts were found to contain also a minor unknown immunoreactive component which we suggest to be some unknown metabolite of melatonin.

Diurnal changes in peripheral melatonin concentration in goats and effects of light or dark interruption.

Diurnal changes in peripheral plasma concentrations of melatonin (MLT) and effects of 2 hr light exposure during the nighttime and 2 hr dark exposure during the daytime on MLT concentrations in goats were examined in natural lighting condition. Two breeds of goats, the Saanen goat as a seasonal breeder at the breeding season, and the Shiba goat as a non-seasonal one, were used while they were at luteal phase. Plasma concentrations of MLT showed a marked diurnal change in both breeds of goats, which was high during the nighttime (20-120 pg/ml) and low during the daytime (<20 pg/ml). Light exposure during the nighttime (for 2 hr) caused an abrupt decline in plasma concentrations of MLT to the basal level; and when the light was turned off, plasma MLT began to increase. On the contrary, 2 hr dark treatment during the daytime did not induced a rapid increase in plasma MLT as observed at the onset of the night. The pattern and the magnitude of the nocturnal elevation in plasma MLT and the responses to the dark or light interruption were virtually identical between the two breeds. These results suggest that the secretion of MLT in goats is driven by the endogenous rhythm, though the lighting stimulus temporarily suppresses the MLT secretion.

Melatonin assays in body fluids.

A variety of methods now exist for the assay of melatonin in body fluids. Their relative merits are compared and the validation of one in particular (RIA) described. Physiological studies of melatonin by RIA have shown probable modulation of its secretion by gonadal steroids. The circadian activity maximum in the dark phase of one of the pineal melatonin synthesizing enzymes, N-acetyltransferase, is reflected in peripheral melatonin levels. Man, like all other species studied so far, has a dark phase rise in circulating melatonin. During the menstrual cycle, melatonin shows a luteal phase rise. Further evidence of pineal rhythmicity is found in seasonal melatonin variations in man. The study of the rhythmic properties of peripheral melatonin in man may provide important information on central nervous function.