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PubMed1991

Human plasma melatonin and urinary 6-sulphatoxy melatonin: studies in natural annual photoperiod and in extended darkness.

OBJECTIVES: The aims of the study were (1) to examine the human plasma melatonin rhythm at the equinoxes and the solstices in the natural photoperiod (at 35 degrees S); (2) to examine melatonin rhythms in the same subjects under extended darkness conditions to expose any suppressive (gating) effects of light at any time of the year; (3) to undertake a rigorous examination of the relationship between plasma melatonin and the urinary metabolite 6-sulphatoxy melatonin at varying times of the year. DESIGN: At the equinoxes and solstices, unrestricted subjects had hourly urine collections followed by venous blood sampling taken under natural light conditions for 24 hours. Following a 24 hour interval, a similar collection regime was performed with subjects held under conditions of extended darkness (5 hours darkness prior to natural sunset and following natural sunrise) for a further 24 hours. SUBJECTS: Groups of four (minimum) to six female volunteers (age range 18-35 years) were studied, who had a normal lifestyle, no history of depression, and were not taking any medication or recently engaged in shiftwork. MEASUREMENTS: The plasma was assayed for melatonin and the urine samples for 6-sulphatoxy melatonin by radioimmunoassay. RESULTS: The onset of natural melatonin secretion was delayed until after sunset at all seasons but was earlier in summer, and not different from the time of sunset in extended darkness. The offset of melatonin secretion under natural conditions occurred at sunrise in autumn and winter but was delayed until after sunrise during spring and summer, particularly in extended darkness. No significant changes in the duration of melatonin secretion were observed between seasons nor between the duration of melatonin secretion under natural photoperiod or extended darkness. The measurement of 6-sulphatoxy melatonin proved to be a close indicator of the phase and amplitude of secretion of plasma melatonin. Both onset and offset times of 6-sulphatoxy melatonin were delayed compared to the times when plasma melatonin was detectable/undetectable. A good correlation exists between the total plasma melatonin secretion and that of 6-sulphatoxy melatonin. CONCLUSIONS: The results suggest evidence for a suppressive (gating) effect of light at dawn only during summer which was associated with a phase advance of the onset of melatonin secretion at this time of year. The lack of a major gating effect of environment light on melatonin secretion, and the unchanging duration of secretion through the year in the normally entrained human, highlight differences between the human and those photoperiodic animal species which breed seasonally. Urinary 6-sulphatoxy melatonin proved to be a good indicator of plasma melatonin levels under rigorous examination and is confirmed as a useful clinical measure.

Adolescent↗

PubMed1997

Investigation into the contractile response of melatonin in the guinea-pig isolated proximal colon: the role of 5-HT4 and melatonin receptors.

1. The interaction of melatonin (N-acetyl-5-methoxytryptamine) with 5-hydroxytryptamine4 (5-HT4) receptors and/or with melatonin receptors (ML1, ML2 sites) has been assessed in isolated strips of the guinea-pig proximal colon. In the same preparation, the pharmacological profile of a series of melatonin agonists (2-iodomelatonin, 6-chloromelatonin, N-acetyl-5-hydroxytryptamine (N-acetyl-5-HT), 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT)) was investigated. 2. In the presence of 5-HT1/2/3 receptor blockade with methysergide (1 microM) and ondansetron (10 microM), melatonin (0.1 nM-10 microM), 5-HT (1 nM-1 microM) and the 5-HT4 receptor agonist, 5-methoxytryptamine (5-MeOT: 1 nM-1 microM) caused concentration-dependent contractile responses. 5-HT and 5-MeOT acted as full agonists with a potency (-log EC50) of 7.8 and 8.0, respectively. The potency value for melatonin was 8.7, but its maximum effect was only 58% of that elicited by 5-HT. 3. Melatonin responses were resistant to atropine (0.1 microM), tetrodotoxin (0.3 microM), and to blockade of 5-HT4 receptors by SDZ 205,557 (0.3 microM) and GR 125487 (3, 30 and 300 nM). The latter antagonist (3 nM) inhibited 5-HT-induced contractions with an apparent pA2 value of 9.6 GR 125487 antagonism was associated with 30% reduction of the 5-HT response maximum. Contractions elicited by 5-HT were not modified when melatonin (1 and 10 nM) was used as an antagonist. 4. Like melatonin, the four melatonin analogues concentration-dependently contracted colonic strips. The rank order of agonist potency was: 2-iodomelatonin (10.8) > 6-chloromelatonin (9.9) > or = N-acetyl-5-HT (9.8) > or = 5-MCA-NAT (9.6) > melatonin (8.7), an order typical for ML2 sites. In comparison with the other agonists, 5-MCA-NAT had the highest intrinsic activity. 5. The melatonin ML1B receptor antagonist luzindole (0.3, 1 and 3 microM) had no effect on the concentration-response curve to melatonin. Prazosin, an alpha-adrenoceptor antagonist possessing moderate/ high affinity for melatonin ML2 sites did not affect melatonin-induced contractions at 0.1 microM. Higher prazosin concentrations (0.3 and 1 microM) caused a non-concentration-dependent depression of the maximal response to melatonin without changing its potency. Prazosin (0.1 and 1 microM) showed a similar depressant behaviour towards the contractile responses to 5-MCA-NAT. 6. In the guinea-pig proximal colon, melatonin despite some structural similarity with the 5-HT4 receptor agonist 5-MeOT, does not interact with 5-HT4 receptors (or with 5-HT1/2/3 receptors). As indicated by the rank order of agonist potencies and by the inefficacy of luzindole, the most likely sites of action of melatonin are postjunctional ML2 receptors. However, this assumption could not be corroborated with the use of prazosin as this 'ML2 receptor antagonist' showed only a non-concentration-dependent depression of the maximal contractile response to both melatonin and 5-MCA-NAT. Further investigation with the use of truly selective antagonists at melatonin ML2 receptors is required to clarify this issue.

Animals↗

PubMed2004

Melatonin-mediated regulation of human MT(1) melatonin receptors expressed in mammalian cells.

In mammals, the pineal hormone melatonin activates G protein-coupled MT(1) and MT(2) melatonin receptors. Acute exposure of recombinant MT(1) and MT(2) melatonin receptors to supraphysiological concentrations of melatonin differentially regulates these two receptors with the MT(2), but not the MT(1), exhibiting rapid desensitization and internalization. In the present study, we sought to determine whether prolonged exposure to supraphysiological and physiological concentrations of melatonin desensitized and/or internalized the MT(1) melatonin receptor. Using a Chinese hamster ovary (CHO) cell line stably expressing MT(1)-FLAG or transiently expressing MT(1)-green fluorescent protein (GFP) melatonin receptors, we found that prolonged exposure (8h) to supraphysiological concentrations of melatonin (100 nM) significantly increased the number of MT(1) melatonin receptors and decreased the affinity (K(i)) of melatonin for competition for 2-[125]iodomelatonin. A similar treatment also desensitized the MT(1) melatonin receptor-mediated stimulation of [(35)S]GTPgammaS binding, but did not internalize the receptor. In contrast, prolonged exposure to a concentration of melatonin mimicking nocturnal levels (400 pM) did not affect the number of MT(1) melatonin receptors, the affinity for melatonin, or the functional sensitivity of the receptor. We conclude that in vivo endogenous melatonin does not significantly affect the functional sensitivity of MT(1) melatonin receptors, however, exogenous melatonin taken therapeutically at doses above physiological levels could desensitize the receptor thereby affecting physiological responses mediated following activation of MT(1) melatonin receptors.

Animals↗

PubMed1989

Diurnal variations in melatonin binding sites in the hamster brain: impact of melatonin.

The distribution of 125I-melatonin binding sites in the male Syrian hamster brain was recorded at 3 times over a 24 h period. The binding in the hypothalamus, hippocampus, medulla-pons and midbrain of the hamsters varied significantly over the 24 h period with different patterns and phases. No such variations were observed in the parietal cortex. Daily morning (10.00 h) or late afternoon (18.00 h) injections of melatonin for 28 days markedly increased the serum concentrations of melatonin at all times recorded. Serum concentrations of testosterone were significantly lower in animals injected with melatonin in the late afternoon than in the untreated controls; no such decrease was observed in animals injected in the morning despite the continuously elevated levels of circulating melatonin. The daily melatonin injections did not significantly affect 125I-melatonin binding in the hypothalamus, parietal cortex and medulla-pons. In the midbrain, 125I-melatonin binding decreased regardless of the time of injection. In the hippocampus, morning melatonin injections caused a marked decrease in 125I-melatonin binding at all times recorded whereas melatonin injected in the late afternoon led to a decrease in 125I-melatonin binding at 10.00 h only. These results indicate diurnal variations in 125I-melatonin binding sites in discrete brain areas of the golden hamster, persisting despite prolonged duration of elevated levels of circulating melatonin. The differential effects of timed melatonin injections on the hippocampal 125I-melatonin binding sites are positively correlated with the counter-antigonadal response produced by morning melatonin injections.

Animals↗

PubMed2000

Melatonin regulates the mRNA expression of the mt(1) melatonin receptor in the rat Pars tuberalis.

The pars tuberalis (PT) of the pituitary is a major neuroendocrine target site for melatonin as it contains a large number of high-affinity melatonin receptors. We have previously shown that melatonin autoregulates the density of its own receptors in the PT. However, whether melatonin regulation includes mRNA expression in vivo is unclear. In the present study we have used quantitative in situ hybridization to (1) follow the daily profile of mt(1) mRNA expression in the rat PT and (2) investigate whether mt(1) mRNA expression could be regulated in vivo by melatonin. We found clear diurnal variations of mt(1) mRNA expression that persist in constant darkness. We also showed, on pinealectomized animals, that the rhythmic pineal melatonin secretion is necessary for the expression of these daily variations. In a second step, we studied the effect of an acute suppression of endogenous melatonin synthesis on mt(1) melatonin receptors by applying a 1-hour light pulse during the night. We found that light induced a dramatic increase in mt(1) mRNA which was totally prevented by a melatonin injection showing that the acute effect of melatonin on the receptor mRNA is strongly inhibitory. A light pulse applied to animals with a chronic absence of melatonin was ineffective showing that light only affects melatonin receptors via the light-induced plasma melatonin suppression. Altogether our results show that melatonin regulates mt(1) melatonin receptor mRNA expression. However, this regulation seems to be complex: acute changes in plasma melatonin concentration regulate negatively the gene transcription, even if the daily endogenous nocturnal melatonin peak seems a prerequisite for variations in its receptor expression.

Animals↗

PubMed1993

Prolonged exposure to melatonin leads to time-dependent sensitization of adenylate cyclase and down-regulates melatonin receptors in pars tuberalis cells from ovine pituitary.

In photoperiodic mammals, seasonal cycles of growth and reproduction are cued by changes in the duration of the nocturnal profile of secretion of the pineal hormone melatonin. To investigate the likely mode of action of this hormone on target tissues, the effect of prolonged treatment with melatonin on the sensitivity of the adenylate cyclase (AC) system was examined in primary cultures of ovine pars tuberalis (PT) cells. When cells were exposed to melatonin (100 pM or 1 microM) for 16 h, and the hormone was then removed by a series of washes, basal production of cAMP was elevated over that observed in cells not treated with melatonin. Moreover, the rate of accumulation of cAMP after stimulation with forskolin (1 microM) was markedly enhanced in cells previously treated with melatonin compared to that in untreated controls. This sensitization by melatonin of the basal and forskolin-stimulated responses developed gradually and was half-maximal after approximately 8 h of exposure. There was no significant difference between the sensitizing effects of the two melatonin concentrations used. Treatment with melatonin for 24 h reduced the total amount of specific [125I]iodomelatonin binding in PT cell membranes by 30-50%. However, over the same period there was no reduction in the ability of a maximal (1 microM) concentration of melatonin to inhibit forskolin-stimulated cAMP production, indicating the presence of an excess capacity of melatonin receptors in cultured PT cells. Nevertheless, treatment with melatonin for 16 h did result in a 10-fold increase in the IC50 for the inhibition by melatonin of forskolin-stimulated cAMP production. The enhancement of cAMP production after prolonged treatment with melatonin was not masked by the inclusion of isobutylmethylxanthine (1 mM) during the subsequent challenge with forskolin, suggesting that sensitization was not due to a reduction in the activity of cAMP-phosphodiesterase. In control cells, aluminium fluoride caused an inhibition of forskolin-stimulated cAMP production. Prolonged treatment with melatonin abolished the inhibitory effect of aluminium fluoride, suggesting that treatment with melatonin caused a shift in the net balance between the G-protein-mediated stimulatory and inhibitory influences on the AC system. The sensitization of AC was not blocked by the inclusion of cycloheximide (10 micrograms/ml) during prolonged exposure to melatonin, suggesting that de novo protein synthesis is not a requirement for this effect of the hormone. These results constitute the first demonstration of an independent action of melatonin on ovine PT cells that is dependent upon the duration of the endocrine stimulus.(ABSTRACT TRUNCATED AT 400 WORDS)

1-Methyl-3-isobutylxanthine↗

PubMed1994

Influence of acute melatonin treatment and light on the circadian melatonin rhythm in the Djungarian hamster.

The present study tested the hypothesis that acute melatonin treatment affects the biological clock mechanism controlling the circadian melatonin rhythm. Adult Djungarian hamsters were raised in LD 16:8 (lights-on from 0300 to 1900 hr). Pineal and serum melatonin concentrations were assessed at specific clock times during the subsequent LD cycle (day 1) and afterwards in hamsters that remained in constant darkness (day 2), to assess the effects of treatment on the circadian melatonin rhythm. In untreated controls (n = 76), melatonin increased in the pineal and in circulation within the first hour after lights-off (day 1) or subjective night (day 2); melatonin concentrations remained elevated for up to 7 hr. A single melatonin injection (5 micrograms/0.2 ml saline, s.c.), administered in the morning (0900 hr, n = 80) or late afternoon (1600 hr, n = 74), transiently increased in circulation but not in the pineal gland. In constant darkness (day 2), the circadian melatonin rhythm in both the pineal gland and circulation was the same as that in untreated controls. No significant differences were found among the three groups in the mean concentration during the day or night; the rise, fall, peak time, peak amplitude, and duration of increased melatonin were also unchanged. The ability of melatonin to override the effect of a light pulse on the circadian melatonin rhythm was also tested. The 5-min light pulse at night (2300-2305 hr of day 1, n = 64) delayed the rise and shortened the duration of increased melatonin in both pineal gland and serum by as much as 3 hr (day 2); these light effects were more pronounced on the pineal than on the serum melatonin rhythm. Injection with melatonin prior to the light pulse (5 micrograms/0.2 ml saline at 2245 hr, n = 64) failed to alter the inhibitory effects of light on the circadian melatonin rhythm. These data suggest that the biological clock mechanism controlling the circadian melatonin rhythm in the Djungarian hamster in long days is responsive to the phase-shifting effects of light, but resistant to the influence of acute melatonin treatment.

Analysis of Variance↗

PubMed1996

Changes in pineal indoleamines in rats after single melatonin injections: evidence for a diurnal sensitivity to melatonin.

We recently determined that melatonin stimulated serotonin (5-HT) secretion from rat pineal glands by increasing 5-HT release from the pinealocytes (microM melatonin concentrations) and by inhibiting 5-HT uptake in the pineal sympathetic nerve endings (mM melatonin concentrations). The present study investigated whether a single melatonin injection could alter the content of indoleamines in the rat pineal gland, as well as its possible dependence on the daytime of administration. Melatonin (150 micrograms/kg) was i.p. injected at 8 time points (11.00 h, 14.00 h, 17.00 h, 20.00 h, 23.00 h, 02.00 h, 05.00 h and 08.00 h) to rats kept in 12:12 h light:dark cycle (lights on at 07.00 h). Melatonin injections in the afternoon (17:00 h) and late in the nighttime (02.00 h and 05.00 h) decreased pineal 5-HT content 90 min later. The levels of 5-hydroxyindoleacetic acid (5-HIAA) were also decreased 90 min after the melatonin treatment at 14.00 h, 17.00 h and 02.00 h. The effect of melatonin on 5-HT content was a long-fasting effect (still evident after 180 min) only when injected at 02.00 h, whereas 5-HIAA levels were found to be decreased 180 min after melatonin treatment at 14.00 h and 23.00 h. No changes in these compounds were detected 240 min after melatonin treatment. Moreover, melatonin did not change 5-hydroxytryptophan levels at any of the daytime points studied. By contrast, 90 min after the injection of melatonin at 20.00 h, an increased content of pineal N-acetylserotonin was observed. This effect of melatonin could be mediated through a phase alteration of the pineal N-acetyltransferase activity rhythm by acting on the suprachiasmatic clock, although a direct melatonin effect on the pineal rhythmic function cannot be excluded. The effects of the hormone on 5-HT and 5-HIAA contents agree with previous findings on the inhibitory effect of pharmacological doses of melatonin on pineal 5-HT uptake, which presumably would result in a decreased intraneuronal content of 5-HT and its acid metabolite. These data point to an acute regulatory action of exogenous melatonin on the pineal melatonin synthesis pathway which seems to be limited to two daytime phases; the afternoon-early evening period and the second half of the night.

5-Hydroxytryptophan↗

PubMed1997

Studies on the vasoconstrictor action of melatonin and putative melatonin receptor ligands in the tail artery of juvenile Wistar rats.

1. In this study we compared the vasoconstrictor activity of melatonin in rat isolated tail artery using two different recording systems, the Halpern pressure myograph and the Halpern-Mulvany wire myograph, with the view to determining a reliable method for obtaining pharmacological data on vascular melatonin receptors. In addition, we characterized the melatonin receptor in this preparation, using analogues of melatonin, and examined the activity of various naphthalenic derivatives with biological activity in non-vascular models of melatonin receptors. 2. Using the Halpern pressure myograph, cumulative addition of melatonin (0.1 nM to 1 microM) produced direct vasoconstriction (19.3+/-6.4% reduction in lumen diameter, n=5) in five of 11 pressurized segments, with pEC50 of 9.14+/-0.17. Similarly, non-cumulative application of melatonin caused vasoconstriction (19.7+/-4.6% reduction in lumen diameter, n=7) in seven of 20 preparations examined with pEC50 of 8.74+/-0.26. The selective alpha2-adrenoceptor agonist, UK-14304 (5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline bitartrate), produced vasoconstriction in all 'melatonin-insensitive' preparations. 3. Melatonin (0.1 nM to 1 microM) failed to elicit isometric contractions of tail artery segments in the Halpern wire myograph, but produced concentration-dependent potentiation of electrically-evoked, isometric contractions (maximum effect of 150-200% enhancement) when applied either noncumulatively (seven of seven preparations) or cumulatively (four of seven preparations). The pEC50 value of melatonin (non-cumulative) was 8.50+/-0.10 (n=7) which was not different from that obtained in the pressure myograph. All further experiments were conducted using a non-cumulative protocol against electrically-evoked, isometric contractions. 4. Based on the pEC50 values for the melatonin analogues examined, the pharmacological profile for the enhancement of electrically-evoked contractions was 2-iodomelatonin > 6-chloromelatonin > or = (-)-AMMTC > or = S21634 > or = melatonin > or = S20098 > S20242 > or = S20304 > 6-hydroxymelatonin > S20932 > (+)-AMMTC > N-acetyl-5-HT. Our data suggests the vascular receptor belongs to the MEL1-like subtype. All the indole-based analogues of melatonin, 2-iodomelatonin, (-)-AMMTC, (+)-AMMTC, S20932, 6-chloromelatonin, 6-hydroxymelatonin and N-acetyl-5-HT, behaved as full agonists. All the naphthalenic derivatives examined, S21634, S20098, S20242 and S20304 behaved as partial agonists relative to melatonin. 5. The naphthalenic-based antagonists, S20928 and S20929, did not modify electrically-evoked, isometric contractions of the tail artery, but produced a parallel, rightward displacement of the melatonin concentration-response curve. Based upon the effect of 1 microM S20928 and S20929, the estimated pK(B) values for these antagonists were 7.18+/-0.25 (n=4) and 7.17+/-0.25 (n=5), respectively. 6. We demonstrated that enhancement of electrically-evoked, isometric contractions of the rat isolated tail artery (using the Halpern-Mulvany wire myograph) is a simple and reproducible model for assessing the activity of putative agonists, partial agonists and antagonists at vascular melatonin receptors. Pharmacological characterization of the receptor suggests the presence of a MEL1-like subtype.

Animals↗

PubMedDate not supplied

Regional and diurnal studies of melatonin and melatonin binding sites in the duck gastro-intestinal tract.

Since melatonin and putative melatonin receptors can be detected in a variety of peripheral tissues, direct endocrine and paracrine actions of melatonin on the physiological functions of different organ systems in response to internal and external stimuli probably exist. As an extension of our earlier work on the pharmacological characterization of 2-[125I]iodomelatonin binding sites in the duck jejunum, the regional and diurnal variations of melatonin and putative melatonin receptors of different segments of the duck gastro-intestinal tract were studied in an attempt to understand the role of melatonin in the physiology of the digestive system. Although no significant effects of diurnal variation and pinealectomy on the regional distribution of melatonin were observed, significant regional variations of melatonin levels were detected with decreasing levels as follows: colon > oesophagus, caecum > duodenum, jejunum > ileum. The densities of melatonin binding sites showed a significant variation between different intestinal regions at either mid-light or mid-dark, with the following descending order: ileum, jejunum > duodenum, colon > caecum > oesophagus. Analysis of the distribution of melatonin binding sites in the wall of the intestine revealed maximal binding in the mucosa and minimal binding in the muscular layers of the jejunum. Similar results were obtained for other intestinal regions as revealed by autoradiography. No significant changes in the affinities of melatonin binding sites were detected between different regions and tissue layers of the alimentary canal. Moreover, the densities and affinities of melatonin binding sites among different regions of the gut exhibited no significant diurnal variations. The demonstration of regional variations in melatonin levels and the density of melatonin binding sites along the gastro-intestinal tract, with a concentration of the putative melatonin receptors in the mucosal layer, suggests a possible direct action of melatonin in the regulation of fluid/electrolyte transport and nutrient absorption in the gut.

Analysis of Variance↗

PubMed1998

Physiological exposure to melatonin supersensitizes the cyclic adenosine 3',5'-monophosphate-dependent signal transduction cascade in Chinese hamster ovary cells expressing the human mt1 melatonin receptor.

Here, we report the effects of short exposure to melatonin on the human mt1 (h mt1) melatonin receptor-mediated signaling in Chinese hamster ovary (CHO) cells, and the consequences of an exposure that resembles the physiological pattern of melatonin release on cAMP-mediated signal transduction. Short exposure (10 min) of h mt1 melatonin receptors to melatonin (400 pM) inhibited forskolin-stimulated cAMP formation, cAMP-dependent protein kinase activity, and phosphorylation of the cAMP response element-binding protein. However, treatment of mt1-CHO cells with melatonin in a manner that closely mimics the in vivo activation of melatonin receptors (i.e. 400 pM melatonin for 8 h to mimic darkness) resulted in a supersensitization of the cAMP-dependent signal transduction cascade during the period of withdrawal (i.e. 16 h without melatonin to mimic the light cycle of a diurnal photoperiod). During the period of withdrawal, forskolin induced a time-dependent (1-16 h) increase in cAMP formation (approximately 200% of control cells). This effect of melatonin was dependent on the presence of the h mt1 melatonin receptor, as no potentiation of forskolin-induced cAMP formation was observed in CHO cells transfected only with the neomycin resistance plasmid. The time-dependent increase in forskolin-stimulated cAMP levels resulted in a potentiation of cAMP-dependent protein kinase activity 1 h after withdrawal (approximately 130% of control cells; P < 0.05) and in the number of cells containing the phosphorylated form of cAMP response element-binding protein (approximately 75% of cells at 1 and 16 h compared with 30% in control cells; P < 0.05). An increase in the undissociated state (G alphabetagamma) of Gi proteins may underlie this phenomenon as demonstrated by the increase in pertussis toxin-catalyzed ADP-ribosylation of G proteins (217 +/- 48% of control; P < 0.05) after melatonin withdrawal. This increase in the ribosylation was not due to an up-regulation of Galpha(i) protein, as no significant change in Galpha(i) protein levels occurred at this time. We demonstrated that activation of the h mt1 melatonin receptor in a manner that resembles the physiological pattern of melatonin exposure alters signaling, as potentiation of cAMP-mediated signal transduction events is observed after hormone withdrawal. The CHO cells expressing the human melatonin receptor may provide an in vitro cellular model in which to investigate the putative signaling mechanisms leading to gene regulation by melatonin.

Animals↗

PubMed1998

Inconsistent suppression of nocturnal pineal melatonin synthesis and serum melatonin levels in rats exposed to pulsed DC magnetic fields.

The purpose of these experiments was to determine whether the exposure of rats at night to pulsed DC magnetic fields (MF) would influence the nocturnal production and secretion of melatonin, as indicated by pineal N-acetyltransferase (NAT) activity (the rate limiting enzyme in melatonin production) and pineal and serum melatonin levels. By using a computer-driven exposure system, 15 experiments were conducted. MF exposure onset was always during the night, with the duration of exposure varying from 15 to 120 min. A variety of field strengths, ranging from 50 to 500 microT (0.5 to 5.0 G) were used with the bulk of the studies being conducted using a 100 microT (1.0 G) field. During the interval of DC MF exposure, the field was turned on and off at 1-s intervals with a rise/fall time constant of 5 ms. Because the studies were performed during the night, all procedures were carried out under weak red light (intensity of <5 microW/cm2). At the conclusion of each study, a blood sample and the pineal gland were collected for analysis of serum melatonin titers and pineal NAT and melatonin levels. The outcome of individual studies varied. Of the 23 cases in which pineal NAT activity, pineal melatonin, and serum melatonin levels were measured, the following results were obtained; in 5 cases (21.7%) pineal NAT activity was depressed, in 2 cases (8.7%) studies pineal melatonin levels were lowered, and in 10 cases (43.5%) serum melatonin concentrations were reduced. Never was there a measured rise in any of the end points that were considered in this study. The magnitudes of the reductions were not correlated with field strength (i.e., no dose-response relationships were apparent), and likewise the reductions could not be correlated with the season of the year (experiments conducted at 12-month intervals under identical exposure conditions yielded different results). Duration of exposure also seemed not to be a factor in the degree of melatonin suppression. The inconsistency of the results does not permit the conclusion that pineal melatonin production or release are routinely influenced by pulsed DC MF exposure. In the current series of studies, a suppression of serum melatonin sometimes occurred in the absence of any apparent change in the synthesis of this indoleamine within the pineal gland (no alteration in either pineal NAT activity or pineal melatonin levels). Because melatonin is a direct free radical scavenger, the drop in serum melatonin could theoretically be explained by an increased uptake of melatonin by tissues that were experiencing augmented levels of free radicals as a consequence of MF exposure. This hypothetical possibly requires additional experimental documentation.

Animals↗

PubMed2006

Melatonin enhances alkaline phosphatase activity in differentiating human adult mesenchymal stem cells grown in osteogenic medium via MT2 melatonin receptors and the MEK/ERK (1/2) signaling cascade.

The goals of this study were to determine (a) if melatonin enhances human adult mesenchymal stem cell (hAMSC) differentiation into osteoblasts as assessed by measuring alkaline phosphatase (ALP) enzyme activity, and (b) identify potential signal transduction pathways that mediate this process. ALP activity significantly increased in hAMSCs following a 10-day incubation in osteogenic medium, relative to hAMSCs incubated in basal growth medium alone. Melatonin (50 nm), added in combination with the osteogenic medium, significantly increased ALP activity relative to osteogenic medium alone. Co-exposure of hAMSCs to osteogenic medium supplemented with melatonin and either pertussis toxin or the melatonin receptor antagonists, luzindole or 4P-PDOT (MT2 receptor selective), inhibited the melatonin-induced increase in ALP activity, indicating the involvement of melatonin receptors, in particular, MT2 receptors. Assessment of melatonin receptor function following exposure to osteogenic medium containing either vehicle or melatonin produced dichotomous results. That is, if the differentiation of hAMSCs into an osteoblast was induced by osteogenic medium alone, then 2-[125I]-iodomelatonin binding and melatonin receptor function increased. However, examination of melatonin receptor function following chronic melatonin exposure, an exposure that resulted in a 50% enhancement in ALP activity, revealed that these receptors were desensitized. This was reflected by a complete loss in specific 2-[125I]-iodomelatonin binding as well as melatonin efficacy to inhibit forskolin-induced cAMP accumulation. Further characterization of the mechanisms underlying melatonin's effects on these differentiation processes revealed that MEK (1/2) and ERK (1/2), epidermal growth factor receptors, metalloproteinase and clathrin-mediated endocytosis were essential while PKA was not. Our results are consistent with a role for melatonin in osteoblast differentiation. If so, then, the decrease in plasma melatonin levels observed in humans during late adulthood may further enhance susceptibility to osteoporosis.

Adult↗

PubMed2006

Effect of caffeine intake 12 or 24 hours prior to melatonin intake and CYP1A2*1F polymorphism on CYP1A2 phenotyping by melatonin.

Earlier evidence suggests that melatonin is almost exclusively metabolised by CYP1A2 and could serve as a probe drug for CYP1A2 phenotyping. However, caffeine inhibits the metabolism of melatonin by CYP1A2 and dietary caffeine could be a potential confounder for the measurement of CYP1A2 activity with melatonin. We undertook a 3-phase cross-over study in 12 healthy volunteers to examine whether caffeine (200 mg single dose), taken 12 hr or 24 hr prior to melatonin intake, would affect the results of CYP1A2 phenotyping results as assessed by a spot sample melatonin concentration 1.5 hr after intake of 6 mg of melatonin orally. In addition we examined the influence of the CYP1A2*1F polymorphism on the phenotyping results by combining the present material with another 12 persons from a previous study. Caffeine, co-administered 12 or 24 hr prior to melatonin intake, did not have any significant effect on the 1.5 hr melatonin concentration (P=0.086 for ANOVA), but in two volunteers about 4 times increase in melatonin concentration was observed after caffeine intake 12 hr (but not 24 hr) before phenotyping with melatonin. Also, individuals homozygous for the CYP1A2*1A allele had clearly higher 1.5 hr melatonin concentration compared with the *1F/*1F or the *1F/*1A genotypes. Abstinence from caffeine for 24 hr prior to melatonin intake should be enough to overcome the possible confounding effect of caffeine on the CYP1A2 phenotyping with melatonin. Also, melatonin may be a sensitive probe to detect phenotypic differences with regard to CYP1A2*1F polymorphism. Melatonin might be, thus, advantageous for CYP1A2 phenotyping compared to the standard probe caffeine.

Administration, Oral↗

PubMed1980

Melatonin given in the morning prevents the suppressive action on the reproductive system of melatonin given in late afternoon.

Melatonin administered to female hamsters kept under light:dark cycles of 14:10 (in hours) normally suppresses reproductive processes only if the indoleamine is administered later than 6.5 h after the beginning of the photoperiod. In the present study, we investigated the influence of morning (11.00 h) injections of melatonin on the reproductive inhibitory effects of afternoon (17.00 h) melatonin injections. Adult female hamsters were exposed to light daily from 06.00 to 20.00 h. The animals were divided into the following experimental groups: group 1, injected with vehicle at both 11.00 and 17.00 h; group 2, injected with vehicle at 11.00 h and 25 microgram melatonin at 17.00 h; group 3, injected with 1 mg melatonin at 11.00 h and 25 microgram melatonin at 17.00 h; group 4, injected with 1 mg melatonin at 11.00 h and vehicle at 17.00 h. Control animals injected with vehicle at both 11.00 and 17.00 h had normal 4-day estrous cycles throughout the 8 weeks of the experiment. 100% of the animals injected with vehicle in the morning and melatonin in the afternoon became acyclic within 7 weeks. However, if the afternoon injections of melatonin were preceded by morning injections of the indoleamine, the animals continued to exhibit normal estrous cycles. Also, hamsters injected with melatonin at 11.00 h and vehicle at 15.00 h had normal estrous cycles throughout the study. At the conclusion of the experiment, the uterine weights and plasma prolactin levels in the animals that received vehicle at 11.00 h and malatonin at 17.00 h were depressed compared to those in the vehicle-vehicle injected controls. Again, the morning injections of melatonin prevented the afternoon injections of melatonin from decreasing either the uterine weights or the plasma prolactin levels. It is concluded that the morning injections of melatonin either down-regulated the melatonin receptors or decreased their number and thereby rendered afternoon injections of melatonin incapable of inhibiting reproductive processes.

Animals↗

PubMed1984

Development and validation of a melatonin radioimmunoassay using radioiodinated melatonin as tracer.

A new radioimmunoassay (RIA) using [125I]-melatonin as tracer for determination of melatonin in biological fluids has been developed. Melatonin antisera were raised in rabbits by immunization with bovine thyroglobulin conjugate of N-acetyl-5- methoxytryptophan . Two high-affinity and specific antisera were obtained. Unlike in previous studies melatonin was radioiodinated directly. Iodo-Gen was used as the oxidant. Radioiodinated melatonin was purified by TLC for use in RIA. Melatonin was extracted from plasma, serum and urine samples (1 ml) with chloroform. Using the extraction the sensitivity of the RIA method was 18 fmol/ml of original sample. Plasma, serum and urine extracts diluted parallelly with synthetic melatonin in RIA. HPLC analysis of plasma and serum extracts showed only one immunoreactive peak co-eluting with synthetic melatonin. Majority of urine immunoreactivity co-eluted with synthetic melatonin, but 7-23% contaminating immunoreactivity was also observed. Daytime values for rat plasma, human serum and urine melatonin were 30-60, 20-40 and 50-130 fmol/ml and the respective night values were 160-300, 180-370 and 230-470 fmol/ml. Thus a characteristic diurnal rhythm of melatonin was observed in all cases. The urinary excretion of immunoreactive melatonin during the day was 3-9 and during the night 11-28 pmol/h. Thus we have developed a specific and valid RIA method for the determination of plasma and serum melatonin. Despite the incomplete specificity of the RIA for urine determinations, a clear diurnal rhythm for urine melatonin was observed. The distinct advantage of the utilization of [125I]melatonin as tracer is that the costly and cumbersome scintillation counting can be avoided.

Animals↗

PubMed1998

Effects of two melatonin analogues, S-20098 and S-20928, on melatonin receptors in the pars tuberalis of the rat.

By using quantitative autoradiography, we studied the effects of two drugs related to melatonin on the 2-(125)I-melatonin binding in the pars tuberalis (PT) of rats. The drugs tested were two naphthalenic analogues of melatonin, S-20098 (N-[2-(7-methoxy-1-naphthyl) ethyl] acetamide), an agonist, and S-20928 (N-[2-(1-naphthyl) ethyl] cyclobutyl carboxamide), a putative antagonist. Melatonin (s.c. and i.p.), S-20098 (s.c.), and S-20928 (i.p.) were injected 4 hr before sacrifice. Acute administration of both melatonin and S-20098 decreased melatonin receptor density. In contrast, the putative antagonist S-20928, at a low dose (1 mg/kg), was ineffective on melatonin receptors. It neither affected the 2-(125)I-melatonin specific binding observed in the control group nor did it prevent the decrease in binding induced by melatonin when injected 5 min before the hormone. At a high dose (10 mg/kg), S-20928 totally blocked the effect of melatonin on melatonin receptor density and induced a decrease in binding capacity as melatonin did when injected alone. These results indicate that in the rat pars tuberalis, the melatonin agonist, S-20098, is able to down-regulate melatonin receptors, whereas S-20928 seems to behave as a partial agonist.

Acetamides↗

PubMed1999

Long-term exposure of hypothalamic explants to melatonin alters the release of gonadotrophin releasing hormone and the density of melatonin binding sites in the pars tuberalis of the male mink (Mustela vison).

To investigate the action of melatonin on the reproductive system, the effect of prolonged versus short-term exposure to melatonin on the release of gonadotrophin releasing hormone (GnRH) was examined in hypothalamic explants of male mink sacrificed in July, September or November. Mediobasal hypothalamic (MBH) explants including the pars tuberalis (PT) were incubated for 1 night with or without melatonin (10(-8) M) for 8 hr or 16 hr and the release of GnRH was then measured. The next day, the explants were incubated further but in a melatonin free buffer, and the release of GnRH was measured with increasing time. Half of the July and September explants had melatonin binding sites quantified by autoradiography. In November, a 16-hr exposure to melatonin induced a significant increase in the release of GnRH during the night, compared with control or 8-hr melatonin exposure. This increase persisted for at least 45 min after the withdrawal of melatonin, suggesting a stimulatory effect of melatonin on the synthesis of GnRH; this effect was apparent in July, September and November. In September, the density of melatonin binding in the PT was significantly lower in the explants incubated for 16 hr with melatonin, compared with those incubated for 8 hr. Thus, in vitro, a long exposure to melatonin, mimicking a single long night, stimulates the release and synthesis of GnRH in parallel with a decrease in the density of melatonin binding in the PT. These effects seem to depend heavily on the duration of exposure to melatonin.

Animals↗