Presence Of Melatonin Research Articles

Chronic hypoxia inhibits the antihypertensive effect of melatonin on pulmonary artery

Exposure of animals to chronic hypoxia induces pulmonary vascular remodeling leading to pulmonary hypertension. Melatonin, the principal hormone of the pineal gland, is known to have an inhibitory effect on rat vascular reactivity. This study examined the effect of chronic hypoxia on the influence of melatonin on the vasoreactivity of the pulmonary artery. The inhibitory effect of melatonin on the phenylephrine-induced constriction in normoxia-adapted rings (101.5 ± 4% versus 82.2 ± 4%) in the presence or absence of melatonin, respectively) was lost following chronic hypoxic treatment (100.2 ± 4% versus 102.2 ± 2%) and this effect was independent of the endothelium. Melatonin also significantly enhanced the relaxant response to acetylcholine of the pulmonary arterial rings from normoxic rats (34.76 ± 5.67% versus 53.82 ± 4.736%) in the absence or presence of melatonin, respectively). In contrast, melatonin had no significant effect (21.71 ± 1.37% versus 23.51 ± 6.891%) on the relaxant response to acetylcholine of the pulmonary arterial rings from chronic hypoxia-adapted rats. Pre-treatment with melatonin (10 − 4 M) showed no significant effect on the vasorelaxation by the nitric oxide donor; sodium nitroprusside (10 − 7 –10 − 5 M). The melatonin-induced changes were blocked by the melatonergic-receptor antagonist luzindole (2 × 10 − 6 M). The results from our study confirm the presence of melatonergic receptors on the pulmonary trunk of rats and also suggest that the modulatory role of melatonin on the vasoreactivity of pulmonary trunk does not involve the nitric oxide pathway. Most importantly, our results show that development of pulmonary hypertension in rats is associated with the loss of the vasorelaxant influence of melatonin.

The effect of melatonin and structural analogues on the lipid peroxidation of triglycerides enriched in ω-3 polyunsaturated fatty acids

The lipid peroxidation of triglycerides enriched in polyunsaturated fatty acids was investigated by photoemission techniques and the TBARS assay. Butylated hydroxytoluene, 5-OH-tryptophan and N-acetylserotonin inhibited light emission and TBARS formation in a concentration dependent manner. However, it was enhanced in the presence of melatonin and 5-methoxytryptamine and was dependent on its concentration. The total relative luminic units were found to be lower in those systems incubated in the presence of butylated hydroxytoluene, N-acetylserotonin or 5-OH-tryptophan; this decreased proportionally to the concentration of the compound tested. The order of inhibition was 5-OH-tryptophan > N-acetylserotonin > butylated hydroxytoluene with the following IC50 values: 0.65, 6.5 and 9.0 mM respectively. The free-radical scavenging activity of the indole derivatives was also analyzed by the DPPH method, and the results indicate that 5-OH-tryptophan, and N-acetylserotonin exhibited a dose-dependent free-radical scavenging ability at all of the tested concentrations. Thus, at 10 μM concentration a decrease of 84.71% and 73.50% of initial DPPH was observed, compared to 51.00% of BHT. Melatonin and 5-methoxytriptamine decreased the initial concentration of DPPH only 1.85% and 5.0%, respectively. The possible formation of N 1-acetyl- N 2 formyl-5-methoxykynuramine (AFMK) during lipid peroxidation of triglycerides enriched in PUFAs with cumene hydroperoxide in the presence of melatonin was also analyzed.

Melatonin attenuates the development of antinociceptive tolerance to delta-, but not to mu-opioid receptor agonist in mice

The effects of melatonin (Mel) on the development of tolerance to antinociceptive actions induced by mu- and delta-opioid receptor agonists were determined in male Kunming mice. In the mouse tail-flick tests, selective mu and delta receptor agonists were repeatedly administered to mice supraspinally (intracerebroventricularly, i.c.v.) in the absence or presence of melatonin. Administration of endomorphin-1 (EM-1, a mu-opioid receptor agonist) or deltorphin I (del I, a delta-opioid receptor agonist) twice daily for 4 days produced antinociceptive tolerance compared with vehicle controls. Co-administration with melatonin prevented the development of tolerance to deltorphin I analgesia, and this effect was dose dependent. However, melatonin did not affect the development of antinociceptive tolerance to endomorphin-1. Additionally, the attenuation of deltorphin I tolerance by melatonin was reduced by chronic treatment with luzindole (luz), a selective antagonist on the MT 2 receptor subtype. Taken together, these data suggest that melatonin interferes with the neural mechanisms involved in the development of tolerance to delta-opioid agonist analgesia via its receptor.

Melatonin and its synthesizing enzymes (arylalkylamine N‐acetyltransferase‐like and hydroxyindole‐O‐methyltransferase) in avian eggs and early embryos

The presence of melatonin and the enzymes (transcripts and activities) involved in its synthesis, i.e. arylalkylamine N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT), was investigated in the eggs and early embryos of Japanese quail at Hamburger-Hamilton stages 1-10. Melatonin was present in the egg yolk (approximately 70 pg/g) and albumen (approximately 20 pg/g). The average content of melatonin was approximately 416 pg/egg. AA-NAT and HIOMT transcripts were present in the oocytes, blastoderms, and ovarian follicles. AA-NAT-like and HIOMT activities were detected in quail egg yolk. The activity of AA-NAT in yolk was comparable with that found in the pineal gland when calculated per milligram of yolk or pineal gland, but was significantly lower when re-calculated per milligram of protein in the yolk or pineal gland. AA-NAT-like activity was also identified in the ovarian follicles. Low HIOMT activity was detected in yolk, but not in the ovarian follicle. Both enzymes were essentially absent from early embryos although some residual activities, probably of yolk origin, were present in the stage 1 embryo. Melatonin and all the constituents needed for its synthesis (serotonin, AA-NAT and HIOMT activities) are contained within the avian yolk and could be used by the embryo from the very beginning of its development. The role of extrapineal melatonin in early avian development may be in protecting the embryo from the action of free radicals formed during intensive embryonic metabolism and/or it may participate (together with serotonin) in a 'diffuse neuroendocrine system' acting at early developmental stages, before differentiation of the nervous system.

Melatonin is a phytochemical in olive oil

Given the numerous observations regarding the positive effects of olive oil consumption and the presence of melatonin in edible plants, we addressed for the first time the question of melatonin determination in virgin olive oil. All the extra virgin olive oil registered designation of origins from Spain and commercial samples of refined olive and sunflower oil were used. Immunoprecipitation and ELISA were combined for melatonin determination. Melatonin is present in olive oil at higher levels in extra virgin olive oil than in refined olive or sunflower oil samples. We concluded that melatonin is part of the phytochemical profile of the olive oil. Particularly, extra virgin olive oil had almost double the melatonin contents of the other refined oils analysed. Thus, melatonin may account for the healthy effects of the Mediterranean diet in which olive oil is the main source of fat.

Melatonin induces neuritogenesis at early stages in N1E‐115 cells through actin rearrangements via activation of protein kinase C and Rho‐associated kinase

Melatonin increases neurite formation in N1E-115 cells through microtubule enlargement elicited by calmodulin antagonism and vimentin intermediate filament reorganization caused by protein kinase C (PKC) activation. Microfilament rearrangement is also a necessary process in growth cone formation during neurite outgrowth. In this work, we studied the effect of melatonin on microfilament rearrangements present at early stages of neurite formation and the possible participation of PKC and the Rho-associated kinase (ROCK), which is a downstream kinase in the PKC signaling pathway. The results showed that 1 nm melatonin increased both the number of cells with filopodia and with long neurites. Similar results were obtained with the PKC activator phorbol 12-myristate 13-acetate (PMA). Both melatonin and PMA increased the quantity of filamentous actin. In contrast, the PKC inhibitor bisindolylmaleimide abolished microfilament organization elicited by either melatonin or PMA, while the Rho inhibitor C3, or the ROCK inhibitor Y27632, abolished the bipolar neurite morphology of N1E-115 cells. Instead, these inhibitors prompted neurite ramification. ROCK activity measured in whole cell extracts and in N1E-115 cells was increased in the presence of melatonin and PMA. The results indicate that melatonin increases the number of cells with immature neurites and suggest that these neurites can be susceptible to differentiation by incoming extracellular signals. Data also indicate that PKC and ROCK are involved at initial stages of neurite formation in the mechanism by which melatonin recruits cells for later differentiation.

Lipid–protein modifications during ascorbate‐Fe2+ peroxidation of photoreceptor membranes: protective effect of melatonin

The rod outer segment (ROSg) membranes are essentially lipoprotein complexes. Rhodopsin, the major integral protein of ROSg, is surrounded by phospholipids highly enriched in docosahexaenoic acid (22:6 n3). This fluid environment plays an important role for conformational changes after photo-activation. Thus, ROSg membranes are highly susceptible to oxidative damage. Melatonin synthesized in the pineal gland, retina and other tissues is a free radical scavenger. The principal aim of this work was to study the changes in the ROSg membranes isolated from bovine retina submitted to nonenzymatic lipid peroxidation (ascorbate-Fe2+ induced), during different time intervals (0-180 min). Oxidative stress was monitored by increase in the chemiluminescence and fatty acid alterations. In addition we studied the in vitro protective effect of 5 mm melatonin. The total cpm originated from light emission (chemiluminescence) was found to be lower in those membranes incubated in the presence of melatonin. The docosahexaenoic acid content decreased considerably when the membranes were exposed to oxidative damage. This reduction was from 35.5 +/- 2.9% in the native membranes to 12.65 +/- 1.86% in those peroxidized during 180 min. In the presence of 5 mm melatonin we observed a content preservation of 22:6 n3 (23.85 +/- 2.77%) at the same time of peroxidation. Simultaneously the alterations of membrane proteins under oxidative stress were studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Loss of protein sulfhydryl groups and increased incorporation of carbonyl groups were utilized as biomarkers of protein oxidation. In membranes exposed to Fe2+ -ascorbate, we observed a decrease of protein thiols from 50.9 +/- 3.38 in native membranes to 1.72 +/- 2.81 nmol/mg of protein after 180 min of lipid peroxidation associated with increased incorporation of carbonyl groups into proteins from 7.20 +/- 2.50 to 12.50 +/- 1.12 nmol/mg of protein. In the SDS-PAGE we observed a decrease in the content of all the proteins, mainly rhodopsin, as a consequence of peroxidation. Melatonin, prevent both lipid peroxidation and protein oxidation.

The cerebrospinal fluid contributes to the presence of melatonin in the sheep brain

The cerebrospinal fluid contributes to the presence of melatonin in the sheep brain

Melatonin Concentration in Plasma, Pineal Gland and Duodenum of Pregnant Rats and their Fetuses after Melatonin and Phenytoin Administration

Melatonin (MEL) is involved in regulation of variety of physiological processes. Beside the pineal gland, the presence of MEL was confirmed in the gastrointestinal tract (GIT), where it may exert multiple actions. We investigated the effects of a single MEL administration of 0.4, 4.0 and 40.0 μg MEL/ml of tap water on MEL levels in rat plasma, pineal gland and duodenum. In addition, we studied the effect of a prolonged administration of MEL, an anticonvulsant drug phenytoin (PHT) and a combination of MEL+PHT, on MEL concentrations in pregnant rats and their fetuses. Single MEL administration of 4.0 and 40.0 μg MEL/ml resulted in a dose-related increase of MEL concentration in the rat plasma and duodenum but in a decrease of MEL in the pineal gland. After a prolonged MEL administration, MEL levels in duodenal tissues were significantly lower in control group and group treated with PHT as compared to animals, where MEL and PHT were administered simultaneously. A significant increase in MEL concentration was recorded in the intestine of fetuses taken from mothers treated with MEL and MEL + PHT. Our results indicate a dose-related absorption of MEL from tap water to GIT tissues of pregnant rats and their fetuses, where MEL may increase the protection of the intestinal mucose.

The oxidative effects of melatonin, the content of citrate and the activity of aconitate hydratase in the rat liver in toxic hepatitis

When melatonin was injected into the liver of rats with toxic hepatitis, there was a reduction in the rate of free radical processes and in the mobilization of the antioxidative system, as estimated by the parameters of biochemiluminescence and the level of α-tocopherol, which is likely to be accounted for by the antioxidative effect of this hormone. The administration of melatonin to intact animals causes a reduction in the activity of aconitate hydratase and an increase in the level of citrate, which reflects the enhancement of the antioxidative potential as it promotes the decreased hydroxyl radical formation in the Fenton reaction. Metatonin-induced elevations in citrate levels and changes in the activity of aconitate hydratase were observed in the liver of animals with toxic hepatitis as compared with those not receiving the hormone, which seems to be associated with the less mobilization of the antioxidative system in the presence of melatonin that is able to produce an antioxidative effect.

Protective effect of melatonin on ascorbate‐Fe2+ lipid peroxidation of polyunsaturated fatty acids in rat liver, kidney and brain microsomes: a chemiluminescence study

Melatonin (N-acetyl-5-methoxytryptamine), the main secretory product of the pineal gland, is a free radical scavenger that has been found to protect against lipid peroxidation in many experimental models. In the present study the effect of melatonin on lipid peroxidation of long chain polyunsaturated fatty acids located in rat liver, kidney and brain microsomes was determined using gas chromatography and a chemiluminescence assay. In vitro assays showed that after incubation of rat liver, kidney or brain microsomes in an ascorbate-Fe++ system, at 37 degrees C for 180 min, the total cpm originated from light emission (chemiluminescence) was found to be lower in those membranes incubated in the presence of melatonin. The incubation of rat liver, kidney or brain microsomes in the presence of ascorbate-Fe2+ resulted in lipid-peroxidation of membranes as evidenced by light emission and decrease of docosahexaenoic acid 22:6 n-3 and arachidonic acid 20:4 n-6. In the presence of melatonin (0.5, 1.0, 1.5 mm), light emission percent inhibition of microsomes was: (liver - 3.33, 9.98, 39.40) (kidney - 46.79, 61.88, 68.36) and (brain - 33.36, 28.89, 43.32). Not all fatty acids were equally protected after the addition of melatonin to the incubation medium. Our results indicate a selective protection of C20:4 n6 and C22:6 n3 by melatonin during non-enzymatic lipid peroxidation of rat liver, kidney and brain microsomes.

MTNR1A melatonin receptors in the ovine premammillary hypothalamus: day-night variation in the expression of the transcripts.

Melatonin regulation of reproductive functions in sheep is mediated by action in the premammillary hypothalamus (PMH). The aim of this study was to identify the high-affinity melatonin-receptor subtypes expressed in this structure. To achieve this, we used reverse transcription-polymerase chain reaction (RT-PCR) and developed in situ hybridization techniques (ISH). By using RT-PCR, we detected a band corresponding to the MTNR1A melatonin-receptor cDNA in the PMH as well as in the pars tuberalis (PT). On the opposite, MTNR1B melatonin-receptor transcripts were not detected using degenerate primers in any of the structures considered, confirming the lack of expression of this receptor subtype in sheep. The expression of MTNR1A mRNA was further confirmed in the PMH by ISH with a 35S-labeled ovine MTNR1A riboprobe. We next investigated the variation in the expression of MTNR1A mRNA between the end of the day and the end of the night (absence and presence of melatonin, respectively). MTNR1A transcript expression was greater at the end of the night than at the end of the day in the PMH. In contrast, MTNR1A mRNA expression was lower at the end of the night than at the end of the day in the PT. No significant variation in the MTNR1A mRNA expression was observed in a more dorsal hypothalamic area. Overall, these results show that MTNR1A transcripts are expressed in the ovine PMH and that their expression follows a diurnal rhythm, which is different from the pattern of expression observed in the PT.

Neuroprotective effects of melatonin against anoxia/aglycemia stress, as assessed by synaptic potentials and superoxide production in rat hippocampal slices.

Melatonin, which plays an important role in circadian rhythm regulation, is highly potent endogenous free radical scavenger and antioxidant. To clarify the neuroprotective effects of melatonin as a free radical scavenger, we recorded changes in synaptic potentials and monitored the generation of superoxide (O)(2)(-) (using hydroethidine) in the CA1 pyramidal layers of rat hippocampal slices exposed to anoxia/aglycemia ('ischemic') stress. Synaptic responses evoked by stimulation of Schaffer collateral/commissural afferents were suppressed by ischemic stress. When the duration of the stress was 8 min, the suppression was reversible, irrespective of the presence or absence of melatonin treatment, while the amount of O(2)(-) generated was reduced by the presence of melatonin. When stress duration was 12 min, the suppression of synaptic responses lasted more than 90 min, but melatonin significantly improved the recovery. The amount of O(2)(-) generated in the 'recirculation' phase after a 12 min ischemic stress was less in the ischemic alone group than in the melatonin-treated group. This probably reflects that the number of viable cells with the ability to generate O(2)(-) had been reduced by the more severe ischemic stress. Other radical scavengers (ascorbic acid and alpha-tocopherol) had similar effects. These results show that melatonin has the potential to protect the functions of neurons against an ischemic insult by reducing O(2)(-) generation.

Melatonin reduces Fenton reaction-induced lipid peroxidation in porcine thyroid tissue.

Free radicals and reactive oxygen species (ROS) participate in physiological and pathological processes in the thyroid gland. Bivalent iron cation (ferrous, Fe(2+)), which initiates the Fenton reaction (Fe(2+) + H2O2 --> Fe(3+) + *OH + OH(-)) is frequently used to experimentally induce oxidative damage, including that caused by lipid peroxidation. Lipid peroxidation is involved in DNA damage, thus indirectly participating in the early steps of carcinogenesis. In turn, melatonin is a well-known antioxidant and free radical scavenger. The aim of the study was to estimate the effect of melatonin on basal and iron-induced lipid peroxidation in homogenates of the porcine thyroid gland. In order to determine the effect of melatonin on the auto-oxidation of lipids, thyroid homogenates were incubated in the presence of that indoleamine in concentrations of 0.0, 0.00001, 0.0001, 0.001, 0.01, 0.1, 0.25, 0.5, 1.0, 2.5, or 5.0 mM. To study melatonin effects on iron-induced lipid peroxidation, the homogenates were incubated in the presence of FeSO(4) (40 microM) plus H2O2 (0.5 mM), and, additionally, in the presence of melatonin in the same concentrations as above. The degree of lipid peroxidation was expressed as the concentration of malondialdehyde + 4-hydroxyalkenals (MDA + 4-HDA) per mg protein. Melatonin, in a concentration-dependent manner, decreased lipid peroxidation induced by Fenton reaction, without affecting the basal MDA + 4-HDA levels. In conclusion, melatonin protects against iron + H2O2-induced peroxidation of lipids in the porcine thyroid. Thus, the indoleamine would be expected to prevent pathological processes related to oxidative damage in the thyroid, cancer initiation included.

Involvement of Bax protein in the prevention of glucocorticoid-induced thymocytes apoptosis by melatonin.

The antiapoptotic effect of melatonin has been described in several systems. In this study, the antagonistic effect of the methoxyindole on dexamethasone-induced apoptosis in mouse thymocytes was examined. Melatonin decreased both DNA fragmentation, and the number of annexin V-positive cells incubated in the presence of dexamethasone. Analysis of the expression of the members of the Bcl-2 family indicated that the synthetic glucocorticoid increased Bax protein levels without affecting the levels of Bcl-2, Bcl-XL, Bcl-XS, or Bak. This effect correlated with an increase in thymocytes bax mRNA levels. Dexamethasone also increased the release of cytochrome C from mitochondria. All of these effects were reduced in the presence of melatonin, which was ineffective per se on these parameters. In addition, the involvement of cAMP on glucocorticoid/melatonin antagonism was examined. Both melatonin and dexamethasone decreased the levels of this nucleotide in mouse thymocytes, indicating that the antagonistic action between both hormones involves a cAMP-independent pathway. In summary, the present results suggest that the antiapoptotic effect of melatonin on glucocorticoid-treated thymocytes would be a consequence of an inhibition of the mitochondrial pathway, presumably through the regulation of Bax protein levels.

Necrotic cell death induced by delta-aminolevulinic acid in mouse astrocytes. Protective role of melatonin and other antioxidants.

Accumulation of delta-aminolevulinic acid (ALA), as it occurs in acute intermittent porphyria (AIP), is the origin of an endogenous source of reactive oxygen species (ROS), which can exert oxidative damage to cell structures. In the present work we examined the ability of different antioxidants to revert ALA-promoted damage, by incubating mouse astrocytes with 1.0 mM ALA for different times (1-4 hr) in the presence of melatonin (2.5 mM), superoxide dismutase (25 units/mL), catalase (200 units/mL) or glutathione (0.5 mM). The defined relative index [(malondialdehyde levels/accumulated ALA) x 100], decreases with incubation time, reaching values of 76% for melatonin and showing that the different antioxidants tested can protect astrocytes against ALA-promoted lipid peroxidation. Concerning porphyrin biosynthesis, no effect was observed with catalase and superoxide dismutase whereas increases of 57 and 87% were obtained with glutathione and melatonin, respectively, indicating that these antioxidants may prevent the oxidation of porphobilinogen deaminase, reactivating so that the AIP genetically reduced enzyme. Here we showed that ALA induces cell death displaying a pattern of necrosis. This pattern was revealed by loss of cell membrane integrity, marked nuclear swelling and double labeling with annexin V and propidium iodide. In addition, no caspase 3-like activity was detected. These findings provide the first experimental evidence of the involvement of ALA-promoted ROS in the damage of proteins related to porphyrin biosynthesis and the induction of necrotic cell death in astrocytes. Interestingly, melatonin decreases the number of enlarged nuclei and shows a protective effect on cellular morphology.

Enhancing effect of melatonin on chemiluminescence accompanying decomposition of hydrogen peroxide in the presence of copper.

The oxidation of melatonin (MEL) using the Cu(II) + H(2)O(2) + HO(-) (the Fenton-like reaction) system was investigated by chemiluminescence (CL), fluorescence, spectrophotometric, and EPR spin trapping techniques. The reaction exhibits CL in the 400-730 nm region. The light emission from the Fenton-like reaction was greatly enhanced in the presence of MEL and was strongly dependent on its concentration. The spectrum measured with cut-off filters revealed maxima at around 460, 500, 580-590, 640-650, and 690-700 nm. The band at 460 nm may be due to the excited cleavage product, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine, whereas the bands at 500, 580-590, 640-650, and 700 nm were similar to those observed for singlet molecular oxygen ((1)O(2)). The effect of reactive oxygen species (ROS) scavengers on the light emission was studied. The CL was strongly inhibited by the (1)O(2) scavengers in a dose-dependent manner; at concentration 1 mM the potency of (1)O(2) scavenging was 5,5-dimethylcyclohexandione-1,3 > methionine > histidine > hydroquinone. The potency of HO(*) scavenging by thiourea, tryptophan, cysteine at concentration 5 mM was 79-94%, by 1 mM glutathione and trolox 75 and 94%, respectively, and by 10 mM cimetidine 18%. Specific acceptors of O(2)(*)(-) such as p-nitroblue tetrazolium chloride and 4,5-dihydroxy-1,3-benzene disulfonic acid (tiron) at concentration 5 mM decreased the CL by 51 and 95%, respectively, whereas superoxide dismutase (SOD) does not reduce the emission at concentration 2.8 U/ml. At higher concentration SOD substantially enhanced the light emission. Addition of 1360 U/ml catalase and 100 microM desferrioxamine strongly inhibited CL (96 and 90%, respectively). The increased generation of (1)O(2) from the Cu/H(2)O(2) system in the presence of MEL was confirmed using the spectrophotometric method based on the bleaching of p-nitrosodimethylaniline and by trapping experiments with 2,2,6,6-tetramethylpiperidine (TEMP) and subsequent electron paramagnetic (EPR) spectroscopy. These findings suggest the increased production of reactive oxygen species (O(2)(*)(-), HO(*), (1)O(2)) from the Fenton-like reaction in the presence of MEL. This means that the hormone is not able to act as classical chain-breaking antioxidant even at low concentration, and may show clear prooxidant activity at higher concentrations. In addition, long-lived carbonyl product of the MEL transformation in the triplet state can also be toxic by transferring its energy to organelles and causing a photochemical process.

Diurnal rhythm of melatonin in young calves and intake of melatonin in milk

AbstractPeripheral concentrations of melatonin in the neonatal mammal are low and the diurnal rhythm is not apparent during the first days or weeks of life. Maternal milk provides a rhythmic although low source of melatonin to the newborn. Diurnal rhythm of serum melatonin was monitored in Friesian calves from birth to 4 weeks of age. Some of the calves under 1 week of age had no clear nocturnal elevation of serum concentrations of melatonin. Thereafter the nocturnal melatonin elevation was apparent in all calves and of the same amplitude as in adult cattle. The effect of oral intake of night-time milk or milk supplemented with low doses of synthetic melatonin was studied during day-time in a group of Ayrshire calves aged 1 to 5 weeks. Two and a half or 3 litres of night-time milk with a mean melatonin concentration of 40 pg/ml, providing 01 to 012 μg melatonin, did not cause any change in the day-time serum concentrations of melatonin, neither did addition of melatonin in doses up to 10 μg per animal. Only the highest dose of melatonin 200 μg in 2 l of milk (01 μg/ml) caused an elevation of day-time serum concentrations of melatonin. These studies show that the pineal gland of a very youg calf is functional and that the concentration of melatonin in blood does not reflect the concentration in milk at the time of feeding. The presence of melatonin in milk suggests, however, its potential significance for the neonate.

Melatonin inhibits growth of diethylstilbestrol-induced prolactin-secreting pituitary tumor in vitro: possible involvement of nuclear RZR/ROR receptors.

Melatonin exerts a marked antiproliferative action in numerous experimentally-induced tumors in vivo as well as in both animal and human cell lines in vitro. However, the mechanisms of oncostatic action of melatonin is not clear, and the involvement of both membrane and nuclear receptors are suggested. Therefore, the aim of this study was to investigate effects of melatonin, and both agonist (CGP 52608), and antagonist (CGP 55644) of RZR/ROR nuclear receptors on the growth of diethylstilbestrol-induced rat prolactin-secreting pituitary tumor cells in vitro. Pituitary tumors were induced by subcutaneous implantation of a single silastic capsule containing 10 mg of diethylstilbestrol in 4-wk-old male Fischer 344 rats. Four months after the implantation of capsules the animals were killed by decapitation, pituitary tumors were aseptically removed, mechanically dispersed, and enzymatically digested with 0.2% collagenase and 0.2% hyaluronidase. The cells (6 x 105 cells/well) were incubated for 24 hr in the presence of melatonin, CGP 52608, CGP 55644 and CGP 55644 plus melatonin (at the concentrations of 107 and 10-9 m) at 37 degrees C in the humidified atmosphere of 95% air and 5% CO2. The group with the addition of solvent only served as control. The growth of cell was measured using the EZ4U system. Statistical analysis was performed using ANOVA followed by LSD test. Both melatonin and CGP 52608 significantly suppressed growth of tumor cells in vitro in both used concentrations. CGP 55644 stimulated growth of tumor cells and blocked the inhibitory effects of melatonin in vitro. Results of the present study as well as other experimental evidence strongly support the hypothesis that both membrane and nuclear receptors are involved in the oncostatic action of melatonin, and indicate that nuclear signalling plays an important role in this process.

Protective melatonin effect on oxidative stress induced by okadaic acid into rat brain.

We studied the effect of melatonin on the oxidative changes produced by the intracerebroventricular (i.c.v.) injection of okadaic acid (200 ng/kg BW) in the Wistar rat. The effects of okadaic acid were evaluated as changes in the quantity of lipid peroxides, reduced glutathione content (GSH) and activity of antioxidative enzymes. Okadaic acid caused lipid peroxidation (5.35 +/- 0.47 micro mol/g tissue in the i.c.v. vehicle group versus 10.14 +/- 0.88 micro mol/g tissue in the okadaic acid group, P < 0.001), GSH consumption (0.115 +/- 0.0065 micro mol/g tissue in the i.c.v. vehicle group versus 0.024 +/- 0.0021 micro mol/g tissue, P < 0.001), and a reduction in the activity of GSH-peroxidase, GSH-reductase and GSH-transferase between 60-80%. All these changes were prevented by pre-injection of 4.5 mg melatonin per kg BW 2 hr before okadaic acid. These findings indicate: (i) okadaic acid induces a status of oxidative stress in the brain, characterized by a high level of lipid peroxidation, decreases in GSH content and diminished activities of antioxidative enzymes, and (ii) melatonin prevents the deleterious effects induced by okadaic acid. In conclusion, the results show the ability of melatonin to modify the neural response to okadaic acid with the protective mechanism likely involving the antioxidative processes of melatonin.