Effects of hypoxia and reoxygenation on the antioxidant defense system of the locomotor muscle of the crab Neohelice granulata (Decapoda, Varunidae).

Crustaceans often occur in areas with variations in oxygen and experience situations known as hypoxia and reoxygenation. Consequences of such situations are increased levels of reactive oxygen species. To avoid oxidative damage intertidal crabs appear to possess an efficient antioxidant defense system (ADS). However, to date, studies have not addressed the strategies that are adopted by the crabs when exposed to hypoxia/reoxygenation cycles. Towards this end we evaluated the ADS and the role of melatonin as an antioxidant in the locomotor muscle of the crab Neohelice granulata under conditions of severe hypoxia and reoxygenation. Total antioxidant capacity against peroxyl radicals and the enzymes superoxide dismutase, catalase, glutathione peroxidase (GPx), and glutathione-S-transferase as well as the key enzyme of glutathione synthesis, glutamate cysteine ligase (GCL), were evaluated. Furthermore, GSH, GSH/GSSG index as well as hemolymph and cellular melatonin levels were evaluated. During hypoxia, increased GPx and GCL activity and decreased GSH and mitochondrial melatonin levels were observed, but during reoxygenation catalase activity increased and cytosolic melatonin levels decreased. It appears that the ADS in the locomotor muscle of N. granulata exert a modulating effect when being confronted with hypoxia and reoxygenation to avoid oxidative stress. During hypoxia, the ADS appear to target GPX activity as well as GSH and mitochondrial melatonin. During reoxygenation, however, evidence suggests that catalase and cytosolic melatonin are involved in the recovery of the locomotor muscle from oxidative damage and the suppression of further damage.

Transcranial bright light exposure via ear canals does not suppress nocturnal melatonin in healthy adults--a single-blind, sham-controlled, crossover trial.

We investigated whether transcranial bright light (TBL) affects nocturnal melatonin and cortisol secretion in sham-controlled crossover trial. Young healthy adults were exposed in random order to 24 minutes of TBL or sham exposure via ear canals at 01:10 h. Saliva and urine samples were collected hourly between 21 h-03 h and 06 h-09 h. There were no significant differences in melatonin or cortisol concentrations between TBL and sham exposures at any sampling point indicating that TBL via ear canals does not suppress nocturnal melatonin secretion. Thus, non-visual effects of TBL are mediated via a pathway not involving melatonin suppression.

The effect of hormone therapy on serum melatonin concentrations in premenopausal and postmenopausal women: a randomized, double-blind, placebo-controlled study.

OBJECTIVES: Melatonin levels decrease physiologically with age, and possibly with the transition to menopause. The plausible influence of hormone therapy (HT) on melatonin is poorly understood. The aim of this randomized, placebo-controlled, double-blind trial was to investigate the effect of HT administration on serum melatonin concentrations in late premenopausal and postmenopausal women. STUDY DESIGN: Analyses were carried out among 17 late premenopausal and 18 postmenopausal healthy women who participated in a prospective HT study in Finland. Serum melatonin was sampled at 20-min (21:00-24:00 h; 06:00-09:00 h) and 1-h (24:00-06:00 h) intervals at baseline and after 6 months with HT or placebo. MAIN OUTCOME MEASURES: Melatonin levels and secretion profile after 6 months of HT compared to placebo. RESULTS: Mean melatonin levels, mean melatonin exposure level (area under curve, AUC) and mean duration of melatonin secretion did not differ after 6 months with HT vs. placebo, irrespectively of the reproductive state. However, in postmenopausal women the melatonin peak time (acrophase) was delayed by 2.4h (2 h 21 min) on average after 6 months with HT vs. placebo (p<0.05). No interaction between time and group was detected when melatonin level was modelled before or after treatment. CONCLUSIONS: Administration of HT to postmenopausal women alters melatonin peak time, but not melatonin levels. Further research on larger clinical samples is needed to better understand the effects of HT on melatonin profile.

Melatonin in perimenopausal and postmenopausal women: associations with mood, sleep, climacteric symptoms, and quality of life.

OBJECTIVE: Melatonin synthesis and secretion are partly modulated by estrogen and progesterone. Changes in melatonin concentrations, possibly related to the menopausal transition, may be associated with climacteric mood, sleep, and vasomotor symptoms. The aims of this study were to compare the serum concentrations of melatonin in perimenopausal and postmenopausal women and to evaluate melatonin's influence on mood, sleep, vasomotor symptoms, and quality of life. METHODS: We analyzed the data of 17 healthy perimenopausal women (aged 43-51 y) and 18 healthy postmenopausal women (aged 58-71 y) who participated in a prospective study. On study night (9:00 pm-9:00 am), serum melatonin was sampled at 20-minute (9:00 pm-12:00 midnight; 6:00-9:00 am) and 1-hour (12:00 midnight-6:00 am) intervals. Questionnaires were used to assess depression (Beck Depression Inventory), anxiety (State-Trait Anxiety Inventory), insomnia and sleepiness (Basic Nordic Sleep Questionnaire [BNSQ]), subjective sleep quality, vasomotor symptoms, and quality of life (EuroQoL). RESULTS: Postmenopausal women had lower nighttime serum melatonin concentrations than perimenopausal women. The duration of melatonin secretion tended to be shorter in postmenopause, whereas melatonin peak time did not differ. Mean melatonin concentrations and exposure levels did not correlate with follicle-stimulating hormone level, estradiol level, body mass index, Beck Depression Inventory score, State-Trait Anxiety Inventory score, BNSQ insomnia score, BNSQ sleepiness score, subjective sleep score, climacteric vasomotor score, or quality of life. In perimenopause, the later is the melatonin peak, the higher is the level of anxiety (P = 0.022), and the longer is the melatonin secretion, the better is the quality of life (P < 0.001). CONCLUSIONS: Longitudinal research is needed to better understand the possible contributory role of menopause in lower melatonin levels.

Effects of melatonin in connection with the antioxidant defense system in the gills of the estuarine crab Neohelice granulata.

Numerous studies have shown that melatonin exerts some influence on the antioxidant defense system (ADS) in vertebrates, but for crustaceans no such effect has been demonstrated till now. However, earlier reports did show a similar profile of daily variations in the ADS of the gills and the melatonin content of the eyestalk in the crab Neohelice granulata and, thus, the aim of this study was to take a closer look at the effects of melatonin in the gill ADS of N. granulata. Gill ADS is to a minor extent modulated by reactive oxygen species (ROS), because only the nonproteic sulfhydryl (NP-SH) content increases (p<0.05) in the presence of hydrogen peroxide (H(2)O(2)). No significant differences (p>0.05) were observed in the melatonin content of the hemolymph between intact and eyestalkless crabs. Gills from intact and eyestalkless crabs injected with physiological saline showed a daily variation in the total peroxyl radical scavenging capacity (TPRSC) (p<0.05) with two peaks, one at the photophase and another at the scotophase. However, in the gills of eyestalkless crabs injected with melatonin (2 x 10(-12)mol crab(-1)), the daily variation in TPRSC values was abolished (p>0.05). This molecule did not change the NP-SH content (p>0.05) in vitro, but decreased (p<0.05) the oxygen consumption in gills when incubated for 120 min. In the in vivo experiments melatonin also decreased (p<0.05) the oxygen consumption in eyestalkless crabs after 390 min. The results suggest that melatonin does not act directly on the ADS of the gills of N. granulata, but decreases the aerobic metabolism possibly involved in variations of tissue ADS.

Long-term postinfarction melatonin administration alters the expression of DHPR, RyR2, SERCA2, and MT2 and elevates the ANP level in the rat left ventricle.

We investigated the effect of 2 wk continuous postinfarction subcutaneous melatonin supply on the expression of the rat left ventricular (LV) dihydropyridine receptor (DHPR), ryanodine receptor (RyR(2)), and sarco-endoplasmic reticulum Ca(2+)-ATPase2 (SERCA2), as they are fundamental proteins in cardiac contractility. The levels of plasma and LV atrial (ANP) and brain natriuretic peptide and melatonin were also measured, as was the expression of LV MT(1) and MT(2) receptors and pineal arylalkylamine N-acetyltransferase. Myocardial infarction (MI) was induced by ligation of the left anterior descending coronary artery and vehicle or melatonin (4.5 mg/kg per day) was administered by subcutaneous osmotic pumps. Echocardiography, real-time quantitative reverse transcription-polymerase chain reaction, and western blotting were used to analyze the samples. Echocardiography revealed that MI induced serious systolic LV dysfunction. The expression of DHPR, RyR(2), and SERCA2 mRNAs was significantly lower in the LVs of melatonin-treated MI rats compared with vehicle-treated rats (P < 0.01 for DHPR and P < 0.05 for RyR(2) and SERCA2). Melatonin also elevated the amount of LV MT(2) receptors to 1.9-fold (P < 0.05) and the concentration of LV ANP to over fivefold (P < 0.05) compared with vehicle rats after MI. Therefore, the results suggest that melatonin may influence the cardiac contractility after MI by regulating the expression of DHPR, RyR(2), and SERCA2, and melatonin receptors, particularly MT(2)s, might contribute to the postinfarction cardioprotective actions of melatonin. Furthermore, the finding of the relationship between melatonin and ANP suggests a novel mechanism for melatonin in protecting the heart after MI.

Daily variation of melatonin content in the optic lobes of the crab Neohelice granulata.

Melatonin is a biogenic amine, known from almost all phyla of living organisms. In vertebrates melatonin is produced rhythmically in the pinealocytes of the pineal gland, relaying information of the environmental light/dark cycle to the organism. With regard to crustaceans only a handful of studies exist that has attempted to identify the presence and possible daily variation of this substance. We set out to investigate whether in the crab Neohelice granulata melatonin was produced in the optic lobes of these animals and underwent rhythmic fluctuations related to the daily light/dark cycle. Our experimental animals were divided into three groups exposed to different photoperiods: normal photoperiod (12L:12D), constant dark (DD), and constant light (LL). The optic lobes were collected every 4 hours over a 24-h period for melatonin quantification by radioimmunoassay (RIA). N. granulata kept under 12 L:12D and DD conditions, showed daily melatonin variations with two peaks of abundance (p<0.05), one during the day and another, more extensive one, at night. Under LL-conditions no significant daily variations were noticeable (p>0.05). These results demonstrate the presence of a daily biphasic fall and rise of melatonin in the eyestalk of N. granulata and suggest that continuous exposure to light inhibits the production of melatonin synthesis.

The effect of myocardial infarction on the synthesis, concentration and receptor expression of endogenous melatonin.

We examined the time course of changes in the synthesis and levels of endogenous melatonin and in the expression of MT(1) and MT(2) melatonin receptors 1 day, 2 and 4 wk after myocardial infarction (MI) in rats. MI was produced by ligation of the left anterior descending coronary artery. Transthoracic echocardiography was performed to characterize structural and functional changes after MI. mRNA levels were measured by real-time quantitative reverse transcription-polymerase chain reaction and proteins by Western blotting. One day after infarction, MI rats had 4.3 times (P < 0.001) higher pineal melatonin synthesis, than sham-operated animals, which was associated with the increased concentration of melatonin in plasma (P < 0.001) and left ventricle (LV) (P = 0.01). The amount of MT(1) receptor protein decreased significantly in MI LVs compared with control LVs 1 day after infarction (P < 0.01), followed by recovery during the next 2 wk. Furthermore, the expression of MT(1) receptor mRNA of the MI LVs was elevated 2 wk after infarction (P < 0.01) compared with control LVs. The amount of MT(2) receptor proteins in MI LVs was higher than in sham-operated LVs 1 day (P < 0.05) and 4 wk (P < 0.01) after MI. In conclusion, melatonin synthesis in the pineal gland increased rapidly in response to the MI, supporting an important role for endogenous melatonin in protecting the heart after MI. The observed changes in the expression of MT(1) and MT(2) receptors suggest that melatonin receptors may be involved in mediating, at least, in part, the protective effects of melatonin in the heart after infarction.

Urinary melatonin: a noninvasive method to follow human pineal function as studied in three experimental conditions.

The aim of this study was to examine whether urinary melatonin, rather than urinary 6-sulfatoxymelatonin (aMT6s), can be used as an indicator of diurnally and seasonally changing melatonin secretion. The subjects (n=15) spent three separate 24-hr periods in a climatic chamber during winter (n=7) and summer (n=8). Blood and urine samples were obtained during each period at 2- to 5-hr intervals. Serum melatonin and urinary melatonin and aMT6s were assayed by radioimmunoassay. The serum melatonin levels increased nearly 10-fold from low daytime to high nocturnal values. The mean nocturnal increase of urinary melatonin was 1.7-fold and that of urinary aMT6s was 4.6-fold. Both urinary melatonin and aMT6s correlated significantly with area under the curve melatonin in serum during the night, during the day and throughout the entire 24-hr observation period in all cases. The ratio between urinary melatonin and aMT6s excretion showed significant diurnal variation, being ninefold higher at 16:00 hr than at 07:00 or at 09:00 hr. The ninefold decrease in the urinary melatonin/aMT6s excretion ratio between the evening and the morning may reflect increased liver metabolism of melatonin during the night. Both urinary melatonin and aMT6s are good indicators of melatonin secretion, but the variation is significantly smaller for the former molecule.

The expression of MT1 and MT2 melatonin receptor mRNA in several rat tissues.

The mechanisms that mediate the various effects of melatonin in mammalian tissues are not always known. Therefore, the aim of this study was to investigate whether MT(1) and MT(2) melatonin receptors are expressed in certain tissues of the rat. The expression of MT(1) and MT(2) melatonin receptor mRNA was determined using a real-time quantitative RT-PCR method. In addition, we examined whether mRNA for either subtype of receptor shows any difference in the expression between midnight and noon, similar to the changes in melatonin concentrations in plasma and tissue samples. MT(1) and MT(2) melatonin receptor mRNAs were found in the rat hypothalamus, retina and small intestine. We also showed a low expression of MT(2) mRNA in the rat liver and heart SA node. In the heart apex and the Harderian gland, no appearance of either of the receptor mRNAs was detectable. A significant difference in the expression of MT(1) mRNA between day and night was found in the hypothalamus. In conclusion, our findings suggest that at least some effects of melatonin are mediated through membrane MT(1) and MT(2) receptors in the hypothalamus, the retina and the small intestine. Down-regulation of receptors might be one reason for the difference in the hypothalamic MT(1) melatonin receptor mRNA expression between midnight and noon. In the liver and the heart SA node, the physiological significance of possible MT(2) receptors remains unclear. According to our negative midnight and noon results in the Harderian gland and heart apex melatonin may exert its effect on these tissues by a non-receptor mechanism.

Endogenous ouabain-like factor (OLF) secretion is modulated by nicotinic mechanisms in rat adrenocortical cells.

This study tested the hypothesis that rat adrenocortical secretion of endogenous ouabain-like factor (OLF) is regulated by nicotinic mechanisms. OLF secreted by dispersed cell suspensions of zona glomerulosa (ZG) and fasciculata/reticularis (ZFR) cells was found to co-elute with authentic ouabain by reverse phase HPLC; OLF concentrations in cell supernatants were measured by radioimmunoassay. Nicotine (10(-6) - 10(-3) M) stimulated significant OLF secretion in rat adrenocortical cells. Acetylcholine (10(-7) - 10(-4) M) and eserine (10(-7) - 10(-3) M) stimulated OLF secretion in ZG cells at lower concentrations and stimulated at higher concentrations. Acetylcholine had no effect on ZFR secretion of OLF, but eserine stimulated OLF secretion. ACTH (10(-8) M) strongly potentiated the OLF stimulatory effect of nicotine in ZG cells; however significant interactions between nicotine and ACTH or angiotensin II on OLF secretion in ZFR cells were not apparent. The ganglionic blockers hexamethonium and mecamylamine further potentiated the effect of nicotine, implicating nicotinic acetylcholine receptors (nAChRs) in regulation of OLF secretion. The alpha7-receptor antagonist methyllycaconitine (MLA) dose-dependently inhibited the effect of nicotine in the ZG cells, and in ZFR cells MLA potentiated nicotine-induced OLF secretion. These data suggest that nicotinic regulation may underlie OLF secretion by rat adrenocortical cells, and strongly suggest presence of functional nicotinic acetylcholine receptors on these cells.

Elevated blood levels of endogenous ouabain-like factor in preterm versus mature newborns at birth.

Compared to healthy adults, elevated levels of endogenous ouabain-like factor (OLF) have already been shown in the cord blood, and a role of OLF in the maintenance of high Na(+) excretion by reducing tubular sodium reabsorption during intrauterine life was suggested. In this study, we aimed to measure OLF cord blood levels of premature and mature newborns to provide further data on the possible physiological significance of this compound in neonates. OLF was assessed in the cord blood of newborns (28-41 weeks) and in the blood of healthy adults using ouabain radioimmunoassay. HPLC was employed to isolate endogenous OLF. Newborns had about twelve times higher OLF levels than healthy adults (41.96 +/- 4.64 vs. 3.1 +/- 1.1 pg/ml, p < 0.001). Further, there was a highly significant correlation (p < 0.004) between maturity and OLF concentration; OLF level increased with gestational age, however there was a rapid drop in its concentration at week 39 (43.26 +/- 7 vs. 35.47 +/- 1.84 pg/ml, p = NS). Further studies are needed to evaluate the physiological relevance of higher OLF in preterm versus mature newborns.

Effect of controlled-release melatonin on sleep quality, mood, and quality of life in subjects with seasonal or weather-associated changes in mood and behaviour.

This study aimed to explore the effects of melatonin on sleep, waking up and well being in subjects with varying degrees of seasonal or weather-associated changes in mood and behaviour. Fifty-eight healthy adults exhibiting subsyndromal seasonal affective disorder (s-SAD) and/or the negative or positive type of weather-associated syndrome (WAS) were randomised to either 2 mg of sustained-release melatonin or placebo tablets 1-2 h before a desired bedtime for 3 weeks. Outcome measures were changes from baseline in sleep quality, sleepiness after waking, atypical depressive symptoms and health-related quality of life by week three. Early morning salivary melatonin concentrations were measured at baseline and treatment cessation in all subjects. Melatonin administration significantly improved the quality of sleep (P=0.03) and vitality (P=0.02) in the subjects with s-SAD, but attenuated the improvement of atypical symptoms and physical parameters of quality of life compared to placebo in the subjects with WAS, positive type.

Important amino acids for the function of the human MT1 melatonin receptor.

Models of G protein-coupled melatonin receptor structure suggest that ligand recognition occurs in a binding pocket formed by transmembrane helices III, V and VII. Constitutively active mutations in G protein-coupled receptors have revealed that transmembrane helix III/intracellular loop 2 interface and transmembrane domain VI are critical regions in receptor activation. In this study, nine site-directed mutants of the human MT1 melatonin receptor were created to test the importance of specific amino acids in these regions in ligand recognition and receptor activation events. We analyzed ligand binding, G protein activation and subcellular localization of MT1 receptors transiently expressed in COS-7 cells. Receptor ELISA was employed to study expression levels of N-terminally HA epitope tagged wild-type and mutant MT1 receptors. Mutations in histidine H195 (His(5.46)) in transmembrane domain V reduced receptor affinity for 2-[125I]iodomelatonin. Several other mutants had diminished expression on the plasma membrane. Amino acids M107 (Met(3.32)) in transmembrane domain III and S280 (Ser(7.46)) in transmembrane domain VII were found not to participate in ligand recognition in human MT1 receptor. Constitutive activity was not obtained with mutations in N124 (Asn(3.49)) or P253 (Pro(6.50)). These mutants failed to bind 2-[125I]iodomelatonin and had reduced expression levels. The need to upgrade current melatonin receptor models has become evident. Several important amino acids for the human MT1 melatonin receptor function were revealed in the current study, with effects of mutations ranging from slightly reduced affinity or efficacy to complete loss of function.