Melatonin and vitamin C increase umbilical blood flow via nitric oxide-dependent mechanisms

Inadequate umbilical blood flow leads to intrauterine growth restriction, a major killer in perinatal medicine today. Nitric oxide (NO) is important in the maintenance of umbilical blood flow, and antioxidants increase NO bioavailability. What remains unknown is whether antioxidants can increase umbilical blood flow. Melatonin participates in circadian, seasonal, and reproductive physiology, but has also been reported to act as a potent endogenous antioxidant. We tested the hypothesis that treatment during pregnancy with melatonin increases umbilical blood flow via NO-dependent mechanisms. This was tested in pregnant sheep by investigating in vivo the effects on continuous measurement of umbilical blood flow of melatonin before and after NO blockade with a NO clamp. These effects of melatonin were compared with those of the traditional antioxidant, vitamin C. Under anesthesia, 12 pregnant sheep and their fetuses (0.8 of gestation) were fitted with catheters and a Transonic probe around an umbilical artery, inside the fetal abdomen. Following 5 days of recovery, cardiovascular variables were recorded during fetal i.v. treatment with either melatonin (n=6, 0.5±0.1 μg/kg/min) or vitamin C (n=6, 8.9±0.4 mg/kg/min) before and after fetal NO blockade with the NO clamp. Fetal treatment with melatonin or vitamin C increased umbilical blood flow, independent of changes in fetal arterial blood pressure. Fetal NO blockade prevented the increase in umbilical blood flow induced by melatonin or vitamin C. Antioxidant treatment could be a useful clinical tool to increase or maintain umbilical blood flow in complicated pregnancy.     

Melatonin rescues cardiovascular dysfunction during hypoxic development in the chick embryo

There is a search for rescue therapy against fetal origins of cardiovascular disease in pregnancy complicated by chronic fetal hypoxia, particularly following clinical diagnosis of fetal growth restriction (FGR). Melatonin protects the placenta in adverse pregnancy; however, whether melatonin protects the fetal heart and vasculature in hypoxic pregnancy independent of effects on the placenta is unknown. Whether melatonin can rescue fetal cardiovascular dysfunction when treatment commences following FGR diagnosis is also unknown. We isolated the effects of melatonin on the developing cardiovascular system of the chick embryo during hypoxic incubation. We tested the hypothesis that melatonin directly protects the fetal cardiovascular system in adverse development and that it can rescue dysfunction following FGR diagnosis. Chick embryos were incubated under normoxia or hypoxia (14% O₂) from day 1 ± melatonin treatment (1 mg/kg/day) from day 13 of incubation (term ~21 days). Melatonin in hypoxic chick embryos rescued cardiac systolic dysfunction, impaired cardiac contractility and relaxability, increased cardiac sympathetic dominance, and endothelial dysfunction in peripheral circulations. The mechanisms involved included reduced oxidative stress, enhanced antioxidant capacity and restored vascular endothelial growth factor expression, and NO bioavailability. Melatonin treatment of the chick embryo starting at day 13 of incubation, equivalent to ca. 25 wk of gestation in human pregnancy, rescues early origins of cardiovascular dysfunction during hypoxic development. Melatonin may be a suitable antioxidant candidate for translation to human therapy to protect the fetal cardiovascular system in adverse pregnancy.     

Lack of effect of melatonin on myometrial electromyographic activity in the pregnant sheep at 138-142 days gestation (term = 147 days gestation)

A 24-h rhythm has been demonstrated in fetal and maternal melatonin plasma concentrations in pregnant sheep in the last third of gestation. Melatonin in the maternal circulation can cross the placenta and is the major source of melatonin in the fetal circulation. Melatonin has been postulated to act as a prostaglandin (PG) synthetase inhibitor in the uterus. PG synthetase inhibitors decrease myometrial contractility. To assess transplacental passage of melatonin and potential influences of melatonin on uterine contractility, we infused melatonin continuously into the maternal jugular vein in seven pregnant sheep at 138-142 days gestation (term in our instrumented animals is 147 days gestation) at three infusion rates for successive 1-h periods during the late morning to late afternoon. There was no change in the total time during which the myometrium was active, as indicated by myometrial electromyographic activity or the myometrial contracture frequency during the 3 h before and after melatonin infusions and for each hour of the infusions. The MCR for melatonin in the ewe was 4128 +/- 410 ml/min (mean +/- SE; n = 7; weight, 50-70 kg). The resting maternal to fetal melatonin concentration ratio was 0.8; this ratio was maintained at 2.28 during melatonin infusion to the ewe at a wide range of maternal melatonin concentrations. Melatonin concentrations in the range of 3-200 times normal had no effect on the maternal plasma PGF2 alpha metabolite concentration, but caused a 40.4% fall in fetal plasma PGE2 (P less than 0.05). We conclude that changes in maternal and fetal plasma melatonin concentrations within the physiological range observed throughout the day do not alter myometrial contractility, but do alter fetal PGs.     

Intracoronary melatonin increases coronary blood flow and cardiac function through β-adrenoreceptors, MT1/MT2 receptors, and nitric oxide in anesthetized pigs

Melatonin is involved in the regulation of the cardiovascular system through the modulation of sympathetic function and the nitric oxide (NO)-related pathway and interaction with MT1/MT2 receptors. However, information regarding its direct actions on coronary blood flow and cardiac function is scarce. This study therefore determined the primary in vivo effect of melatonin on cardiac function and perfusion and the involvement of the autonomic nervous system, MT1/MT2 receptors, and NO. In 35 pigs, melatonin infused into the coronary artery at 70 pg for each mL/min of coronary blood flow while preventing changes in heart rate and arterial pressure increased coronary blood flow, dP/dt(max), segmental shortening, and cardiac output by about 12%, 14%, 8%, and 23% of control values (P < 0.05), respectively. These effects were accompanied by an increase in coronary NO release of about 46% (P < 0.05) of control values. The aforementioned responses were graded in a further five pigs. Moreover, the blockade of muscarinic cholinoreceptors (n = 5) and α-adrenoreceptors (n = 5) did not abolish the observed responses to melatonin. After β(1)-adrenoreceptors blocking (n = 5), melatonin failed to affect cardiac function, whereas β(2)-adrenoreceptors (n = 5) and NO synthase inhibition (n = 5) prevented the coronary response and the effect of melatonin on NO release. Finally, all effects were prevented by MT1/MT2 receptor inhibitors (n = 10). In conclusion, melatonin primarily increased coronary blood flow and cardiac function through the involvement of MT1/MT2 receptors, β-adrenoreceptors, and NO release. These findings add new information about the mechanisms through which melatonin physiologically modulates cardiovascular function and exerts cardioprotective effects.     

Melatonin increases activities of glutathione peroxidase and superoxide dismutase in fetal rat brain

Melatonin is a powerful scavenger of oxygen free radicals. In humans, melatonin is rapidly transferred from the maternal to the fetal circulation. To investigate whether or not maternal melatonin administration can protect the fetal rat brain from radical-induced damage by increasing the activities of antioxidant enzymes, we administered melatonin to pregnant rats on day 20 of gestation. Melatonin (10 mg/kg) was injected intraperitoneally at daytime (14:00 hr) and, to remove the fetuses, a laparotomy was performed at 1, 2, or 3 hr after its administration. We measured the melatonin concentration in the maternal serum and in fetal brain homogenates and determined the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in fetal brain homogenates. Melatonin administration markedly increased melatonin concentrations in the maternal serum and fetal brain homogenates, with peak levels achieved 1 hr after melatonin administration (serum: 538.2+/-160.7 pM/mL; brain homogenates: 13.8+/-2.8 pM/mg protein). Between 1 and 3 hr after melatonin administration, GSH-Px activity in fetal brain homogenates increased significantly (P<0.01). Similarly, SOD activity increased significantly between 1 and 2 hr after melatonin administration (P<0.01). These results indicate that melatonin administration to the mother increases antioxidant enzyme activities in the fetal brain and may thereby provide indirect protection against free radical injury. Thus, melatonin may potentially be useful in the treatment of neurodegenerative conditions that may involve excessive free radical production, such as fetal hypoxia and preeclampsia.     

The role of calcitonin gene-related Peptide in the in vivo pituitary-adrenocortical response to acute hypoxemia in the late-gestation sheep fetus

This study tested the hypothesis that calcitonin gene-related peptide (CGRP) has a role in mediating the in vivo fetal adrenal glucocorticoid response to acute stress. The hypothesis was tested by investigating the effects of fetal treatment with a selective CGRP antagonist on plasma ACTH and cortisol responses to acute hypoxemia in the late-gestation sheep fetus. Under anesthesia, six fetuses at 0.8 of gestation were surgically instrumented with vascular catheters. Five days later, fetuses were subjected to 0.5-h hypoxemia during treatment with either iv saline or a CGRP antagonist, in randomized order, on different days. Treatment started 30 min before hypoxemia and ran continuously until the end of the challenge. Arterial blood samples were collected for plasma ACTH and cortisol measurements (RIA) and blood gas monitoring. CGRP antagonism did not alter basal arterial blood gas or endocrine status. During hypoxemia, similar falls in arterial partial pressure of oxygen occurred in all fetuses. During saline infusion, acute hypoxemia induced significant increases in fetal ACTH and cortisol concentrations. During CGRP antagonism, the pituitary-adrenal responses were markedly attenuated. Correlation of paired plasma ACTH and cortisol values from all individual fetuses during normoxia and hypoxemia showed positive linear relationships; however, neither the slope nor the intercept of the peptide-steroid relationship was affected by CGRP antagonism. These data support the hypothesis that CGRP is involved in the in vivo regulation of fetal adrenocortical steroidogenesis during acute hypoxemia. In addition, the data reveal that CGRP may have a role in the control of other components of the hypothalamo-pituitary-adrenal axis during stimulated conditions in fetal life.     

Melatonin stimulates glutathione peroxidase activity in human chorion

In preeclampsia, placental production of lipid peroxides is abnormally increased, while placental glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities are decreased. Administration of melatonin, a powerful scavenger of oxygen free radicals, also may protect the placenta from free radical-induced damage by increasing the activity of antioxidant enzymes. To test this hypothesis we administered melatonin to pregnant women before they underwent voluntary interruption of pregnancy between 7 and 9 wk of gestation. Melatonin (6 mg) was administered orally at 12:00 hr, and samples of chorion and maternal blood were obtained at the time of the procedure, 1, 2 or 3 hr later. We measured the melatonin concentration in maternal serum and activities of GSH-Px and SOD and levels of melatonin in chorionic homogenates. Melatonin administration was reflected by markedly increased melatonin concentrations in maternal serum and in chorion, with peak levels achieved 1 hr after melatonin administration (serum, 46.87 +/- 10.87 nM/L; chorionic homogenate, 4.36 +/- 1.56 pmol/mg protein). Between 1 and 3 hr after melatonin administration, GSH-Px activity in chorionic homogenates increased significantly (P < 0.001), with peak levels occurring at 3 hr (51.68 +/- 3.22 mU/mg protein per min, 137.3% of GSH-Px activity in untreated control subjects). No significant changes in chorionic SOD activity occurred during the 3-hr post-administration period. These results indicate that exogenous melatonin increases GSH-Px activity in the chorion and thereby may protect indirectly against free radical injury. Melatonin could be useful in treating preeclampsia and possibly other clinical states involving excessive free radical production, such as intrauterine fetal growth retardation and fetal hypoxia.     

Melatonin improves cerebrovascular function and decreases oxidative stress in chronically hypoxic lambs

Chronic hypoxia during gestation and delivery results in oxidative stress and cerebrovascular dysfunction in the neonate. We assessed whether melatonin, a potent antioxidant and potential vasodilator, improves the cerebral vascular function in chronically hypoxic neonatal lambs gestated and born in the highlands (3600 m). Six lambs received melatonin (1 mg/kg per day oral) and six received vehicle, once a day for 8 days. During treatment, biometry and hemodynamic variables were recorded. After treatment, lambs were submitted to a graded FiO₂ protocol to assess cardiovascular responses to oxygenation changes. At 12 days old, middle cerebral arteries (MCA) were collected for vascular reactivity, morphostructural, and immunostaining evaluation. Melatonin increased fractional growth at the beginning and improved carotid blood flow at all arterial PO₂ levels by the end of the treatment (P < 0.05). Further, melatonin treatment improved vascular responses to potassium, serotonin, methacholine, and melatonin itself (P < 0.05). In addition, melatonin enhanced the endothelial response via nitric oxide‐independent mechanisms in isolated arteries (162 ± 26 versus 266 ± 34 AUC, P < 0.05). Finally, nitrotyrosine staining as an oxidative stress marker decreased in the MCA media layer of melatonin‐treated animals (0.01357 ± 0.00089 versus 0.00837 ± 0.00164 pixels/μm², P < 0.05). All the melatonin‐induced changes were associated with no systemic cardiovascular alterations in vivo. In conclusion, oral treatment with melatonin modulates cerebral vascular function, resulting in a better cerebral perfusion and reduced oxidative stress in the neonatal period in chronically hypoxic lambs. Melatonin is a potential therapeutic agent for treating cerebrovascular dysfunction associated with oxidative stress and developmental hypoxia in neonates.     

Chronic hypoxia modulates the function and expression of melatonin receptors in the rat carotid body

Melatonin modulates the carotid chemoreceptor response to chemical stimuli, and chronic hypoxia changes circadian activities and carotid body function. The purpose of this study was to test the hypothesis that chronic hypoxia alters the function and expression of melatonin receptors in the rat carotid body. Effects of melatonin on the carotid responses to hypercapnic acidosis and to hypoxia were determined by spectrofluorometric measurement of cytosolic calcium ([Ca(2+)](i)) in fura-2-loaded type-I (glomus) cells dissociated from carotid bodies obtained from normoxic (Nx) or chronically hypoxic (CH) rats breathing 10% oxygen for 4 wk. In the Nx control, melatonin concentration dependently attenuated the peak [Ca(2+)](i) response to hypercapnic acidosis, whereas it augmented the [Ca(2+)](i) response to cyanide or deoxygenated buffer. Yet, melatonin enhanced the peak [Ca(2+)](i) responses to hypercapnic acidosis or hypoxia in the CH glomus cells. An agonist of melatonin receptors, iodomelatonin also elevated the hypercapnic or hypoxic responses in the CH groups. The melatonin-induced changes in the [Ca(2+)](i) responses were abolished by pretreatment with nonselective mt(1)/MT(2) antagonist, luzindole, and by MT(2) antagonists, 4-phenyl-2-propionamidotetraline or DH97. These findings suggest a functional modulation of melatonin receptors in the glomus cells in chronic hypoxia. To evaluate the level of expression of the melatonin receptors, in situ hybridization study with antisense mt(1) and MT(2) receptor mRNA oligonucleotide probes was performed on the Nx and CH carotid bodies. There were significant increases in the expression of mt(1) and MT(2) receptors in the CH comparing with the Nx group. Taken together, our results suggest an upregulation of the carotid expression of melatonin receptors by chronic hypoxia, which modulates the carotid response to melatonin for the circadian influence on breathing.     

Neuropeptide Y in the sheep fetus: effects of acute hypoxemia and dexamethasone during late gestation

Plasma concentrations of neuropeptide Y (NPY) were measured in pregnant ewes and their fetuses under basal conditions and in response to acute hypoxemia during late gestation. The effects of fetal treatment with dexamethasone on these NPY responses were also examined. Under general anesthesia, 10 Welsh Mountain ewes and their fetuses were chronically instrumented between 117-120 days gestation (dGA; term is approximately 145 dGA) with vascular and amniotic catheters, and an ultrasonic probe around a femoral artery of each fetus. At 124 dGA, five fetuses were continuously infused i.v. with dexamethasone for 48 h at a rate of 1.73 +/- 0.16 microg x kg(-1) x h(-1) while the remaining five fetuses received vehicle at the same rate. At 126 dGA, 45 h from the onset of either infusion, 1 h of materno-fetal hypoxemia was induced by reducing maternal FiO2. During normoxia, maternal plasma NPY concentrations were three times those measured in fetal plasma in both groups. During hypoxemia, PaO2 fell to similar levels in the control and dexamethasone-treated groups in both mothers and fetuses. In control animals, there was a significant increase in the NPY concentration in fetal, but not maternal, plasma during hypoxemia. Fetal treatment with dexamethasone significantly enhanced the fetal NPY response to acute hypoxemia but had no effects on basal NPY levels in the fetal or maternal plasma or on the maternal response to acute hypoxemia. These data show: 1) differences between the maternal and fetal plasma NPY response to maternal inhalation hypoxia; 2) that NPY may play a role in mediating fetal defense responses to acute hypoxemia; and 3) that fetal exposure to glucocorticoids modifies the fetal plasma NPY response to acute hypoxemia.     

Fetal and maternal sheep hypothalamus pituitary adrenal axis responses to chronic binge ethanol exposure during the third trimester equivalent

BACKGROUND: We tested the hypothesis that in utero ethanol exposure results in changes in fetal and maternal adrenocorticotropin (ACTH) and cortisol during the third trimester equivalent, by using a chronically instrumented fetal sheep model. METHODS: Pregnant ewes received saline or ethanol intravenously 3 consecutive days per week from day 109 to day 132 of gestation. Fetal and maternal blood samples were collected on days 118 and 132. RESULTS: Maternal and fetal ACTH and cortisol values increased on days 118 and 132 of gestation in response to ethanol infusions that created blood ethanol concentrations (BECs) that are easily achievable by human drinkers. Peak ACTH and cortisol values were detected 30 to 60 min after peak BECs were achieved. CONCLUSIONS: Chronic ethanol exposure during the third trimester equivalent in sheep resulted in repeated activation of the hypothalamus-pituitary-adrenal axis in both the mother and fetus. Temporally, the patterns of maternal and fetal responses to ethanol infusion were similar. We conclude that ovine maternal ethanol exposure during the third trimester equivalent increases fetal ACTH and cortisol concentrations, hormonal responses that may play a role in mediating alcohol-related birth defects.     

Melatonin enhances the hypoxic response of rat carotid body chemoreceptor

Melatonin attenuates carotid chemoreceptor response to hypercapnic acidosis and may contribute to the effect of circadian rhythms on the chemoreflex. The purpose of this study was to test the hypothesis that melatonin modulates rat carotid chemoreceptor response to hypoxia. To examine the effect of melatonin on the hypoxic response of the chemosensitive cells, cytosolic calcium ([Ca2+]i) was measured by spectrofluorometry in fura-2-loaded type-I (glomus) cells dissociated from rat carotid bodies. Melatonin (0.01-10 nm) did not change the resting Ca2+]i level of the glomus cells but it concentration-dependently increased peak Ca2+]i response to cyanide or deoxygenated buffer. An agonist of melatonin receptors, iodomelatonin also enhanced the Ca2+]i response to hypoxia. The melatonin-induced enhancement of the Ca2+]i response was abolished by pretreatment with nonselective mt1/MT2 antagonist, luzindole, and by MT2 antagonists, 4-phenyl-2-propionamidotetraline or DH97. These findings suggest that melatonin receptors in the glomus cells mediate the effect of melatonin on the chemoreceptor response to hypoxia. In addition, melatonin increased the carotid afferent response to hypoxia in unitary activities recorded from the sinus nerve in isolated carotid bodies superfused with bicarbonate-buffer saline. Furthermore, plethysmographic measurement of ventilatory activities in unanesthetized rats revealed that melatonin (1 mg/kg, i.p.) increased the ventilatory response to hypoxia. Hence, the circadian rhythm of melatonin in arterial blood can modulate the carotid chemoreceptor response to hypoxia. This modulation may be a physiological mechanism involved in the day-light differences in ventilatory activities.     

Melatonin concentration in the umbilical artery and vein in human preterm and term neonates and neonates with acute fetal distress

In order to assess the existence of a rhythmic secretion of melatonin (aMT) in newborns and whether this rhythm is affected by neonatal stress, we studied 112 newborns classified in three groups: normal babies delivered at term, preterm infants born before the 38th week, and babies with fetal distress. Melatonin was measured by RIA in the umbilical artery and vein at the time of birth. Melatonin levels in umbilical arterial and venous blood showed a diurnal rhythm in all groups. Melatonin levels in umbilical cord artery and vein were closely related. Nocturnal melatonin levels were increased in newborns with acute fetal distress in comparison with normal term and preterm neonates. These results suggest that (1) a rhythm of aMT secretion exists in newborns, although it cannot be determined whether this rhythm is of maternal or fetal origin and (2) neonatal stress (acute fetal distress) increases aMT production during the night in comparison with normal term and preterm neonates.     

Peripheral chemoreceptor control of fetal renin responses to hypoxia and hypercapnia

The renin response to hypoxia in late gestation fetal sheep has been well characterized. However, the renin response to asphyxia--the combination of hypoxia and hypercapnia--has not been extensively studied. The purpose of this study was to determine 1) the interaction of hypoxia and hypercapnia in the control of renin secretion in late gestation fetal sheep and 2) the role of peripheral arterial chemoreceptors therein. Chronically catheterized fetal sheep (intact or sinoaortic denervated) were exposed to hypoxia and/or hypercapnia for 30 minutes. Hypercapnia alone had no effect on plasma renin activity or aldosterone but did result in a significant increase in angiotensin II. Hypercapnia combined with hypoxia resulted in a significant increase in renin activity, angiotensin II, and aldosterone. Sinoaortic denervation attenuated the renin and angiotensin II responses to hypercapnia plus hypoxia. The increase in renin and angiotensin II in response to hypercapnia with or without concomitant hypoxia strongly correlated with the magnitude of the decrease in arterial pH in intact fetuses only. Hypoxia alone and in concert with hypercapnia increased mean arterial pressure and decreased heart rate in intact but not sinoaortic denervated fetuses. We conclude that 1) hypercapnia more potently increases plasma renin activity than does hypoxia in late gestation fetal sheep, 2) arterial pH may be the relevant signal perceived by the peripheral arterial chemoreceptors for the control of the renin-angiotensin system during asphyxia, and 3) the cardiovascular response to hypoxia is mediated, in part, by peripheral arterial chemoreceptors.     

Melatonin ameliorates hippocampal nitric oxide production and large conductance calcium-activated potassium channel activity in chronic intermittent hypoxia

Melatonin protects against hippocampal injury induced by intermittent hypoxia (IH). IH-induced oxidative stress is associated with decreases in constitutive production of nitric oxide (NO) and in the activity of large conductance calcium-activated potassium (BK) channels in hippocampal neurons. We tested the hypothesis that administration of melatonin alleviates the NO deficit and impaired BK channel activity in the hippocampus of IH rats. Sprague-Dawley rats were injected with melatonin (10 mg/kg, i.p.) or vehicle before daily IH exposure for 8 hr for 7 days. The NO and intracellular calcium ([Ca2+]i) levels in the CA1 region of hippocampal slices were measured by electrochemical microsenor and spectrofluorometry, respectively. The activity of BK channels was recorded by patch-clamping electrophysiology in dissociated CA1 neurons. Malondialdehyde levels were increased in the hippocampus of hypoxic rats and were lowered by the melatonin treatment. Levels of NO under resting and hypoxic conditions, and the protein expression of neuronal NO synthase (nNOS) were significantly reduced in the CA1 neurons of hypoxic animals compared with the normoxic controls. These deficits were mitigated in the melatonin-treated hypoxic rats with an improved [Ca2+]i response to acute hypoxia. The open probability of BK channels was decreased in the hypoxic rats and was partially restored in the melatonin-treated animals, without alterations in the expression of channel subunits and unitary conductance. Acute treatment of melatonin had no significant effects on the BK channel activity or on the [Ca2+]i response to hypoxia. Collectively, these results suggest that melatonin ameliorates the constitutive NO production and BK channel activity via an antioxidant mechanism against an IH-induced down-regulation of nNOS expression in hippocampal neurons.     

Melatonin protects against the free radical-induced impairment of nitric oxide production in the human umbilical artery

We evaluated melatonin's antioxidative effect on the free radical-induced impairment of nitric oxide production in the human umbilical artery, which may play an important role in fetal hypoxia and ischemia during preeclampsia. Umbilical artery sections with intact endothelium were obtained from healthy pregnant women who were delivered between 37 and 40 wk of gestation. The production of nitric oxide in the umbilical arteries was stimulated by adding L-arginine followed by incubation for 60 min. Nitric oxide concentrations were estimated by measuring nitrite ions (NO2), using high-performance liquid chromatography. Prior to the addition of L-arginine, the segments were treated with hydrogen peroxide (H2O2) alone (1, 10, 100 microM), or were pretreated with either 50 mM mannitol or melatonin (20, 100, 500 microM) before adding H2O2. Changes in L-arginine-induced NO2 production were expressed as a percentage of NO2 production at the end of preincubation. NO2 production was significantly increased by incubating the umbilical artery sections with L-arginine (P<0.01). Treatment with H2O2 significantly reduced L-arginine-induced NO2-production in a concentration-dependent manner (P<0.01). Pretreatment with melatonin significantly increased NO2 production that had been decreased by H2O2 in a concentration-dependent manner (P<0.01). Similarly, pretreatment with mannitol reversed the H2O2-induced reduction in NO2- production (P<0.001). These results indicate that H2O2 may impair nitric oxide synthesis in the endothelium of human umbilical arteries. Melatonin significantly suppresses the H2O2-induced inhibition effect of nitric oxide production, most likely through its ability to scavenge hydroxyl radicals.     

Fetal prolactin levels respond to a maternal melatonin implant

Seasonal PRL changes in adult sheep are controlled by photoperiod. The ability to detect photoperiod is mediated by the pineal gland through melatonin secretion. A rhythm in plasma melatonin has been described in fetal sheep. In this work we investigated whether the fetus responds to changes in circulating levels of melatonin. Fetal plasma PRL was measured every 2 h for 24 h, 7 days after the mother had received a sc Silastic implant containing approximately 1 g melatonin. Control fetuses received an empty implant. Melatonin is slowly released from implants, and it is known to cross the sheep placenta. Fetal plasma melatonin increased 10-fold after the implant. Plasma PRL in fetuses treated with melatonin was a third of that of control fetuses. Fetal plasma PRL concentrations were 56.6 +/- 5.7 ng/ml (mean +/- SE) in control and 18.1 +/- 2.7 ng/ml 7 days after the melatonin implant. We conclude from these data that the ability to respond to melatonin is present in sheep during fetal life.     

Maternal pinealectomy abolishes the diurnal rhythm in plasma melatonin concentrations in the fetal sheep and pregnant ewe during late gestation

We have investigated the effect of pinealectomy of ewes in pregnancy on the presence of the diurnal rhythm in fetal and maternal plasma concentrations of melatonin. Six ewes were pinealectomized between 104 and 118 days of gestation. Fetal and maternal blood samples were collected during 24-h periods between 125 and 140 days of gestation in the pinealectomized ewes and in an intact control (n = 4). There was a significant diurnal rhythm in both fetal and maternal plasma concentrations of melatonin in the control group. In this group, the fetal and maternal plasma melatonin concentrations were significantly higher in the dark (128.4 +/- 6.2 and 192.2 +/- 10.7 pmol/l respectively) than in the light (46.2 +/- 4.2 and 25.8 +/- 2.1 pmol/l respectively). However there was no diurnal rhythm in either the fetal or maternal plasma melatonin concentrations in the pinealectomized group between 125 and 140 days of gestation. In contrast to the control animals, there was also no light-dark difference in the fetal or maternal plasma melatonin concentrations in four pinealectomized animals sampled frequently in the 3-7 days preceding delivery (mean length of gestation 146.5 days). However, in the pinealectomized sheep there was a gradual increase in the combined light-dark fetal plasma melatonin concentrations during late gestation from 27.9 +/- 2.8 pmol/l (at 15-20 days before delivery) to 95.2 +/- 14.1 pmol/l on the day of delivery. We have therefore demonstrated that the maternal pineal is the major source of the diurnal rhythm in maternal and fetal plasma melatonin concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Melatonin enhances pro-inflammatory cytokine levels and protects against Chagas disease

Abstract:  Pro-inflammatory and modulatory cytokines have an essential role in host defense against human and murine Trypanosoma cruzi infection. Control of T. cruzi parasitism during the acute phase of infection is considered to be critically dependent on direct macrophage activation by cytokines. Melatonin has been proposed to regulate the immune system by affecting cytokine production in immunocompetent cells, enhancing the production of several T helper (Th)1 cytokines. The aims of this work were to evaluate in rats, the influences of exogenous melatonin treatment on T. cruzi- infected host's immune responses. With this in mind, several immunological parameters were analyzed, including tumor necrosis factor-α, γ-interferon, interleukin-12, nitric oxide (NO) and macrophage count. The melatonin therapy was provided in one of two different treatment regimens, that is, either beginning 7 days prior to infection or concomitant with the infection. Both treatments triggered an up-regulation of the immune response, with the concomitant treatment being more effective; in this case all cytokines studied, with exception of NO, displayed enhanced concentrations and there was a higher number of peritoneal macrophages, which displayed reduced concentrations under melatonin therapy. We conclude that melatonin plays a pivotal role in up-regulating the Th1 immune response thus controlling parasite replication.     

Melatonin, as an adjuvant-like agent, enhances platelet responsiveness

Abstract:  Melatonin exerts immunomodulatory actions that enhance the magnitude and quality of immune responses specific for certain antigens; this has raised the possibility of using melatonin to design novel vaccine adjuvant systems. The present study investigated the effect of subcutaneous slow-release melatonin implants and subcutaneous melatonin injections on the responsiveness of circulating platelets in sheep after vaccination against Dichelobacter nodosus (A1 and C serotypes), the bacterium that causes ovine footrot, a major cause of lameness in sheep. The experiments were carried out in sheep from a farm located in an area of Mediterranean-type ecosystem. Plasma melatonin levels were determined by radioimmunoassay, sheep platelet aggregation was monitored using an aggregometer and Ca²⁺ mobilization was determined by spectrofluorimetry using fura-2. Administration of melatonin either by implants or subcutaneous injections increased plasma melatonin concentrations, an effect that was found to be greater and more sustained when melatonin was administered via implants. Vaccination per se, as well as melatonin, increased the percentage and rate of platelet aggregation and reduced the lag-time in response to the physiological agonist thrombin, an effect that was found to be significantly greater when melatonin was administered to vaccinated animals. Melatonin enhanced thrombin-evoked Ca²⁺ release and entry and further increased Ca²⁺ mobilization observed in platelets from vaccinated sheep. These observations suggest that the use of melatonin, as a novel adjuvant, induces beneficial effects on platelet function and haemostasis, and opens new perspectives for therapeutic manipulation of immune responses to vaccination.