Effects of Melatonin on the Defense to Acute Hypoxia in Newborn Lambs.

Neonatal lambs, as other neonates, have physiologically a very low plasma melatonin concentration throughout 24 h. Previously, we found that melatonin given to neonates daily for 5 days decreased heart weight and changed plasma cortisol and gene expression in the adrenal and heart. Whether these changes could compromise the responses to life challenges is unknown. Therefore, firstly, we studied acute effects of melatonin on the defense mechanisms to acute hypoxia in the neonate. Eleven lambs, 2 weeks old, were instrumented and subjected to an episode of acute isocapnic hypoxia, consisting of four 30 min periods: normoxia (room air), normoxia after an i.v. bolus of melatonin (0.27 mg kg-1, n = 6) or vehicle (ethanol 1:10 NaCl 0.9%, n = 5), hypoxia (PaO2: 30 ± 2 mmHg), and recovery (room air). Mean pulmonary and systemic blood pressures, heart rate, and cardiac output were measured, and systemic and pulmonary vascular resistance and stroke volume were calculated. Blood samples were taken every 30 min to measure plasma norepinephrine, cortisol, glucose, triglycerides, and redox markers (8-isoprostane and FRAP). Melatonin blunted the increase of pulmonary vascular resistance triggered by hypoxia, markedly exacerbated the heart rate response, decreased heart stroke volume, and lessened the magnitude of the increase of plasmatic norepinephrine and cortisol levels induced by hypoxia. No changes were observed in pulmonary blood pressure, systemic blood pressures and resistance, cardiac output, glucose, triglyceride plasma concentrations, or redox markers. Melatonin had no effect on cardiovascular, endocrine, or metabolic variables, under normoxia. Secondly, we examined whether acute melatonin administration under normoxia could have an effect in gene expression on the adrenal, lung, and heart. Lambs received a bolus of vehicle or melatonin and were euthanized 30 min later to collect tissues. We found that melatonin affected expression of the immediate early genes egr1 in adrenal, ctgf in lung, and nr3c1, the glucocorticoid receptor, in adrenal and heart. We speculate that these early gene responses may contribute to the observed alterations of the newborn defense mechanisms to hypoxia. This could be particularly important since the use of melatonin is proposed for several diseases in the neonatal period in humans.

Deciphering the Function of the Blunt Circadian Rhythm of Melatonin in the Newborn Lamb: Impact on Adrenal and Heart.

Neonatal lambs, as with human and other neonates, have low arrhythmic endogenous levels of melatonin for several weeks until they start their own pineal rhythm of melatonin production at approximately 2 weeks of life. During pregnancy, daily rhythmic transfer of maternal melatonin to the fetus has important physiological roles in sheep, nonhuman primates, and rats. This melatonin rhythm provides a circadian signal and also participates in adjusting the physiology of several organs in preparation for extrauterine life. We propose that the ensuing absence of a melatonin rhythm plays a role in neonatal adaptation. To test this hypothesis, we studied the effects of imposing a high-amplitude melatonin rhythm in the newborn lamb on (1) clock time-related changes in cortisol and plasma variables and (2) clock time-related changes of gene expression of clock genes and selected functional genes in the adrenal gland and heart. We treated newborn lambs with a daily oral dose of melatonin (0.25 mg/kg) from birth to 5 days of age, recreating a high-amplitude melatonin rhythm. This treatment suppressed clock time-related changes of plasma adrenocorticotropic hormone, cortisol, clock gene expression, and functional genes in the newborn adrenal gland. In the heart, it decreased heart/body weight ratio, increased expression of Anp and Bnp, and resulted in different heart gene expression from control newborns. The interference of this postnatal melatonin treatment with the normal postnatal pattern of adrenocortical function and heart development support a physiological role for the window of flat postnatal melatonin levels during the neonatal transition.

Impact of chronodisruption during primate pregnancy on the maternal and newborn temperature rhythms.

Disruption of the maternal environment during pregnancy is a key contributor to offspring diseases that develop in adult life. To explore the impact of chronodisruption during pregnancy in primates, we exposed pregnant capuchin monkeys to constant light (eliminating the maternal melatonin rhythm) from the last third of gestation to term. Maternal temperature and activity circadian rhythms were assessed as well as the newborn temperature rhythm. Additionally we studied the effect of daily maternal melatonin replacement during pregnancy on these rhythms. Ten pregnant capuchin monkeys were exposed to constant light from 60% of gestation to term. Five received a daily oral dose of melatonin (250 µg kg/body weight) at 1800 h (LL+Mel) and the other five a placebo (LL). Six additional pregnant females were maintained in a 14∶10 light:dark cycles and their newborns were used as controls (LD). Rhythms were recorded 96 h before delivery in the mother and at 4-6 days of age in the newborn. Exposure to constant light had no effect on the maternal body temperature rhythm however it delayed the acrophase of the activity rhythm. Neither rhythm was affected by melatonin replacement. In contrast, maternal exposure to constant light affected the newborn body temperature rhythm. This rhythm was entrained in control newborns whereas LL newborns showed a random distribution of the acrophases over 24-h. In addition, mean temperature was decreased (34.0±0.6 vs 36.1±0.2°C, in LL and control, respectively P<0.05). Maternal melatonin replacement during pregnancy re-synchronized the acrophases and restored mean temperature to the values in control newborns. Our findings demonstrate that prenatal melatonin is a Zeitgeber for the newborn temperature rhythm and supports normal body temperature maintenance. Altogether these prenatal melatonin effects highlight the physiological importance of the maternal melatonin rhythm during pregnancy for the newborn primate.

Melatonina inhibe la respuesta de cortisol a ACTH: posible mediación a través del gen reloj Per1 / Melatonin inhibition of the cortisol response to ACTH may be exerted through period circadian protein homolog 1 (Per1)

Background: Circadian cortisol production results from the interaction of the circadian production of ACTH, the autonomic nervous system and intrinsic factors within the gland. An additional regulator is the neuro-hormone melatonin. In human adrenal gland cultures, melatonin inhibited ACTH stimulated cortisol production and Per1 mRNA expression. ACTH actions on the adrenal involve early and late responses. Aim: To investigate the effects of melatonin on the time course of ACTH stimulated cortisol production and of Per1 expression in the lamb adrenal gland. Material and Methods: Adrenal glands and plasma of five newborn lambs were obtained. Adrenal glands were cut in 15 mg explants. Three of these explants were stored for RNA extraction. The rest of explants were using in different culture protocols with ACTH and melatonin. Results: Lambs had an in vivo a circadian variation in plasma cortisol and in adrenal Per1 expression. In vitro, ACTH stimulated an early and late increase in cortisol production and an early increase in Per1 expression reaching a maximum at 3 hours of treatment. Melatonin inhibited the early Per1 response to ACTH without affecting the early ACTH stimulated cortisol production. However, melatonin inhibited the late response of cortisol production to ACTH. Conclusions: The inhibitory actions of melatonin on Per1 response to ACTH may contribute to the inhibitory effects of melatonin on adrenal steroidogenic response to ACTH.

Circadian rhythms in the fetus.

Throughout gestation, the close relationship between mothers and their progeny ensures adequate development and a successful transition to postnatal life. By living inside the maternal compartment, the fetus is inevitably exposed to rhythms of the maternal internal milieu such as temperature; rhythms originated by maternal food intake and maternal melatonin, one of the few maternal hormones that cross the placenta unaltered. The fetus, immature by adult standards, is however perfectly fit to accomplish the dual functions of living in the uterine environment and developing the necessary tools to "mature" for the next step, i.e. to be a competent newborn. In the fetal physiological context, organ function differs from the same organ's function in the newborn and adult. This may also extend to the developing circadian system. The information reviewed here suggests that the fetal circadian system is organized differently from that of the adult. Moreover, the fetal circadian rhythm is not just present simply as the initial immature expression of a mechanism that has function in the postnatal animal only. We propose that the fetal suprachiasmatic nucleus (SCN) of the hypothalamus and fetal organs are peripheral maternal circadian oscillators, entrained by different maternal signals. Conceptually, the arrangement produces internal temporal order during fetal life, inside the maternal compartment. Following birth, it will allow for postnatal integration of the scattered fetal circadian clocks into an adult-like circadian system commanded by the SCN.

High prevalence of vitamin D deficiency in Chilean healthy postmenopausal women with normal sun exposure: additional evidence for a worldwide concern.

OBJECTIVE: To assess the prevalence of vitamin D deficiency in healthy postmenopausal women with normal sun exposure but without vitamin D fortification in their diets. DESIGN: We studied 90 healthy ambulatory women who were residents of Santiago, Chile (latitude 33 degrees S); 30 were premenopausal (32.6 +/- 7.4 y), and 60 were postmenopausal (63.7 +/- 9.7 y). Half of the women were studied during the winter and the other half during the following summer. Each provided a fasting blood sample to measure biochemical parameters, parathyroid hormone, and 25-hydroxyvitamin D and completed a questionnaire to estimate sunlight exposure. A first morning urine sample was collected in postmenopausal women to measure deoxypyridinoline. Various cutoff points of 25-hydroxyvitamin D were used to assess the prevalence of vitamin D deficiency (<9, <15, and <20 ng/mL). RESULTS: All of the women had normal renal and liver parameters. Sunlight exposure was adequate in almost all of the volunteers (93% in both groups, P > 0.05). In postmenopausal women, serum 25-hydroxyvitamin D was less than 9 ng/mL in 12%, less than 15 ng/mL in 40%, and less than 20 ng/mL in 60%, compared with 0%, 13%, and 27%, respectively, in premenopausal women. Deoxypyridinoline was 75% higher during winter than summer (9.8 +/- 2.5 vs 5.6 +/- 1.4 nmol/mmol creatinine, P < 0.0001). CONCLUSIONS: Vitamin D deficiency is very common in Chilean healthy postmenopausal women with normal sun exposure but without vitamin D fortification in their diets. This finding is associated with higher bone resorption during winter time and emphasizes the need to increase vitamin D intake in healthy postmenopausal women.

[Free urinary cortisol is elevated in patients with low-renin essential hypertension]. / Elevación del cortisol urinario en hipertensos esenciales hiporreninémicos.

BACKGROUND: Glucocorticoids play a key role in blood pressure (BP) control and are associated with hypertension in patients with Cushing's syndrome. A number of reports indicate that cortisol (F) may be involved in etiology of essential hypertension (EH). F can bind to the mineralocorticoid receptor, triggering both sodium and water reabsorption in kidney, increase BP and cause renin suppression. AIM: To evaluate urinary free cortisol (UFF) excretion as a potential intermediate phenotype of essential hypertension and correlate F level with plasma renin activity (PRA) and serum aldosterone (SA). PATIENTS AND METHODS: We recruited 132 EH patients and 16 normotensive healthy controls. Blood samples and 24 hours urine were collected for PRA, SA and UFF analysis. Differences in UFF excretion between sexes were normalized by urinary creatinine (Creat) excretion. The upper limit of UFF/Creat was determined in normotensives considering the mean value plus 2 standard deviations. According to this value, subjects were classified as having high or normal UFF. RESULTS: In EH patients and in normotensives, the UFF/Creat was 36.9 +/- 17.0 microg/gr and 30.9 +/- 8.8 microg/gr, respectively. The upper limit was set at 48.5 microg/gr. A high UFF/Creat was found in 20/132 EH (15%) patients and 0/16 normotensive subjects. EH patients with high UFF showed lower PRA levels than patients with normal cortisol levels (0.78 +/- 0.47 vs. 1.13 +/- 0.66 ng/ml x h, respectively, p=0.027) and lower SA values (4.52 +/- 1.65 vs 6.34 +/- 3.37 ng/dl, respectively, p=0.018). There was a negative correlation between UFF and PRA (r=-0.176, p=0.044) and between UFF and SA (r=-0.183, p=0.036). CONCLUSIONS: We have identified a subgroup of EH patients with increased UFF excretion. Patients with the highest UFF showed lower renin and aldosterone levels. These data suggest a potential influence of cortisol in the genesis of hypertension.

Raloxifene is a better antioxidant of low-density lipoprotein than estradiol or tamoxifen in postmenopausal women in vitro.

OBJECTIVE: The oxidation of low-density lipoprotein (LDL) is an important factor in the development of atherosclerosis. The antioxidant activity of some compounds buffers the free radicals generated either endogenously or exogenously, thus decreasing the potential damage mediated by oxidation. Estrogens are potent antioxidants of LDL, in vitro and in vivo, a mechanism that could probably influence the cardioprotection associated with hormone replacement therapy in postmenopause. We conducted an in vitro study of the antioxidant effect on LDL of two selective estrogen receptor modulators, raloxifene (RLX) and tamoxifen (TMX), comparing them with the known antioxidant effect of estradiol (E2 ). DESIGN: LDL was isolated by ultracentrifugation from plasma obtained from 12 healthy, untreated, postmenopausal women. Aliquots containing 0.5 mg of LDL protein were incubated for 4 h with CuSO4 (15 micro M) to induce oxidative stress and with one of the three compounds studied: RLX, TMX, or E2 at doses of 0, 1, 2, 3, 5, 15, 50, and 500 micro M, and 1 and 2 mM. Malonaldehyde (MDA, nmol/mg protein) was measured as a marker of LDL oxidation. RESULTS: E2 induced a dose-dependent decrease in MDA concentration. MDA values decreased significantly, as compared with baseline, starting at a concentration of 2 micro M for RLX and 3 micro M for both, TMX, or E2. The dose necessary to reduce the generation of MDA by 50% was significantly lower for RLX (3.3 micro M, < 0.001) than for E2 (24.6 micro M ) or TMX (35.3 micro M). The area under the curve also showed a higher antioxidant activity for RLX compared with TMX or E2 ( < 0.001). CONCLUSIONS: The in vitro antioxidant activity of RLX is substantially more potent than TMX or E2. This finding, added to the other beneficial effects of the drug in the cardiovascular system, could imply some cardioprotector effect.

[11 beta-hydroxysteroid dehydrogenase type 2 activity in Chilean patients with hypertension]. / Actividad de 11 beta hidroxiesteroide dehidrogenasa tipo 2 en hipertensos chilenos.

BACKGROUND: Half of hypertensive patients with low plasma renin activity have a primary hyperaldosteronism. Among the remaining half, 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta HSD2) deficiency plays an important role. This enzyme catalyzes the conversion of cortisol to cortisone, avoiding the interaction of cortisol with the mineralocorticoid receptor. If the enzyme fails, cortisol will stimulate sodium and water reabsorption and increase blood pressure. AIM: To determine biochemical alterations, suggestive of 11 beta HSD2 deficiency, in low-renin hypertensive patients. PATIENTS AND METHODS: Twenty eight hypertensive patients with a plasma renin activity of less than 0.5 ng/ml/h and with a plasma aldosterone of less than 5 ng/dl were studied. Twenty eight normotensive patients were studied as controls. Serum cortisol (RIA), cortisone (ELISA) and the serum cortisol/cortisone ratio were determined in all of them, between 9 and 10 AM. Measurements were confirmed by high pressure liquid chromatography. The serum cortisol/cortisone ratio was considered abnormal when its Ln (cortisol/cortisone) value was over 2 standard deviations of the mean. RESULTS: Serum cortisol was higher in hypertensive subjects than in controls (11.1 +/- 3.3 and 9.2 +/- 2.8 micrograms/dl, respectively; p < 0.05). No differences were observed in serum cortisone (3.4 +/- 1.3 and 3.7 +/- 1.2 micrograms/dl, respectively). Four hypertensive subjects had an abnormally high Ln (cortisol/cortisone) value (1.86; 1.73; 2.07 and 2.01, considering a normal value of less than 1.61). CONCLUSIONS: Four of 28 hypertensive subjects with low plasma renin activity and aldosterone had biochemical alterations suggestive of 11 beta HSD2 deficiency.