Biomedical subjects

Germaine Escames

Publications and source records attributed to Germaine Escames.

29 records · Page 2Linked to original sources

PubMed2003

Characterization of melatonin high-affinity binding sites in purified cell nuclei of the hamster (Mesocricetus auratus) harderian gland.

In the present paper, binding of melatonin to purified cell nuclei from harderian glands of male and female hamsters was assessed. Binding of 125I-melatonin to cell nuclei fulfills the criteria for binding to a receptor site. Binding kinetics exhibit properties such as dependence on time and temperature as well as reversibility, saturability, high affinity and specificity. The dissociation constants (K(D)) and the number of binding sites (B(max)) for the binding of 125I-melatonin to harderian gland nuclei were 260 +/- 56 pm and 12.2 +/- 0.8 fmol/mg protein in male glands, and 280 +/- 43 pm and 9.8 +/- 0.6 fmol/mg protein in female glands, respectively. Competition experiments showed IC50 values for melatonin of 250 +/- 45 pm and 290 +/- 68 pm in male and female glands, respectively. Other indoleamines such as N-acetylserotonine and 5-metoxytryptamine showed IC50 values in the micromolar range, suggesting that the binding sites are specific for melatonin. Hill analyses of the data show nH values of 0.96-0.98, suggesting the existence of a single class of binding sites. These data indicate that specific 125I-melatonin binding sites exist in the cell nuclei of Harderian glands in male as well as in female hamsters, without significant differences between them. The K(D) and B(max) values obtained from the binding in both sexes correlates well with the concentration of melatonin described in these respective Harderian glands. It is hypothesized that the nuclear binding sites of melatonin here described could be a physiological melatonin receptor, which may be involved in the genomic-dependent antioxidant effects of melatonin on hamster Harderian glands elsewhere reported.

Animals↗

PubMed2003

Specific binding of melatonin to purified cell nuclei from mammary gland of swiss mice: day-night variations and effect of continuous light.

Melatonin binding sites were characterized in the nuclei of mouse mammary glands. The specific binding of 2-125I-iodomelatonin by homogenates of purified mammary gland cell nuclei was found to be rapid, reversible and saturable. Binding of 125I-melatonin exhibited day-night variations with the highest binding affinity observed during the dark period and the lowest affinity at midday. However, when the animals were maintained under continuous light exposure, binding of 125I-melatonin to cell nuclei showed a higher affinity than the daytime values found in animals maintained in normal photoperiod. Scatchard analysis of the binding data revealed the existence of a significant night-day variation in the binding kinetics, compatible with the existence of two classes of binding sites: a high-affinity binding site expressed during the night, with KD = 185 +/- 36 pm and a binding capacity of 6.24 +/- 0.4 fmol/mg protein, and a low-affinity site with KD = 562 +/- 57 nm and Bmax = 7.56 +/- 0.49 pmol/mg protein during the day. Interestingly, after 2 wk of continuous exposure to light, the animals killed at an equivalent midday time showed a significant increase in binding affinity, with KD = 1.43 +/- 0.2 nm and Bmax = 24.75 +/- 3.5 fmol/mg protein. Displacement experiments show an IC50 value compatible with the affinity constants obtained in the saturation experiments. These results indicate that the low-affinity binding site may be saturated by the high levels of melatonin found in the mouse mammary gland, and sustain the hypothesis of a circadian regulation of these melatonin binding sites by the photoperiod.

Animals↗

PubMed2003

Changes in brain amino acids and nitric oxide after melatonin administration in rats with pentylenetetrazole-induced seizures.

We examined the effect of melatonin on brain levels of amino acids and nitric oxide (NO) after pentylenetetrazole (PTZ)-induced seizures in rats. Animals were treated with melatonin (10-160 mg/kg, i.p.) 30 min before PTZ administration (100 mg/kg, s.c.), and were killed 3 hr later. At the dose of 80 mg/kg, melatonin significantly increased the latency (5.7-12.7 min) and decreased the duration (31.2-18.4 s) of the first seizure, reducing PTZ induced mortality from 87.5 to 25%. After kill, brains were removed and neurotransmitters and nitrite levels measured in prefrontal cortex (PF), parieto-temporal cortex (PF), striatum (ST), hippocampus (HP) and brain stem (BS) by high performance liquid chromatography. PTZ treatment increased glutamine levels in all brain areas studied, without changes in glutamate, gamma-amino butyric acid (GABA) and glycine. Aspartate and taurine increased in PF and PT and in HS and PT, respectively. Melatonin administration displayed a dose-dependent effect. At doses of 10-40 mg/kg, melatonin counteracted the PTZ-induced glutamine increase and reduced both glutamate and aspartate levels in the studied areas, with minor changes in GABA and glycine content. At doses of 80 and 160 mg/kg, the levels of glutamine, and glutamate, and to a lesser extent aspartate increased, whereas serine levels did not change. These two doses of melatonin also increased taurine, GABA and glycine in most brain areas studied. Treatment with melatonin (40-160 mg/kg) significantly decreased nitrite content in PT cortex, ST and BS areas of epileptic rats, without changes in the other brain regions. The results suggest that the anticonvulsant property of melatonin involves a modulation of both brain amino acids and NO production.

Amino Acids↗

PubMed2003

Relationship between salivary melatonin levels and periodontal status in diabetic patients.

Among other functions, melatonin exerts both antioxidative and immunoregulatory roles. The indoleamine is secreted in the saliva, although its role into the mouth is not known. Diabetic patients frequently display oral cavity pathologies such as periodontal disease (PD), an inflammatory disease coursing with an increase in free radical production. Thus, we compared the degree of PD and interleukin-2 (IL-2) levels with melatonin concentrations in plasma and saliva of diabetic patients. A total of 43 diabetic patients (20 with type I and 23 with type II diabetes) and 20 age- and sex-matched controls were studied. Dental and medical history of all patients was in accordance with the criteria of the WHO. The periodontal status was evaluated by the Community Periodontal Index (CPI). Plasma and salivary melatonin levels were determined by specific commercial radioimmunoassays, and plasma IL-2 was measured using a commercial enzyme-linked immunosorbent assay kit. Diabetic patients had plasma and saliva melatonin levels of 8.98 +/- 7.14 and 2.70 +/- 2.04 pg/mL, respectively. These values were significantly lower (P < 0.001) than those obtained in plasma and saliva of controls (14.91 +/- 4.75 and 4.35 +/- 0.98 pg/mL, respectively). Plasma and salivary melatonin concentrations show a biphasic response in diabetic patients. Melatonin decreased in patients with a CPI index of 2, and then increased reaching highest levels in patients with a CPI index of 4. By contrast, IL-2 levels decreased from CPI index 1 to 4. The results indicate that, in diabetic patients, the presence of a marked impairment of the oral status, as assessed by the CPI index, is accompanied by an increase in plasma and salivary melatonin. The increase in salivary melatonin excretion may have a periodontal protective role.

Diabetes Mellitus, Type 1↗

PubMed2003

Calreticulin-melatonin. An unexpected relationship.

Increasing evidence suggests that melatonin can exert some effect at nuclear level. Previous experiments using binding techniques clearly showed the existence of specific melatonin binding sites in cell nucleus of rat liver. To further identify these sites, nuclear extracts from rat hepatocytes were treated with different percentages of ammonium sulfate and purified by affinity chromatography. Subsequent ligand blot analysis shows the presence of two polypeptides of approximately 60 and approximately 74 kDa that bind specifically to melatonin. N-Terminal sequence analysis showed that the 60 kDa protein shares a high homology with rat calreticulin, whereas the 74 kDa protein shows no homology with any known protein. The binding of melatonin to calreticulin was further characterized incubating 2-[125I]melatonin with recombinant calreticulin. Binding kinetics show a Kd = 1.08 +/- 0.2 nm and Bmax = 290 +/- 34 fmol.mg protein-1, compatible with other binding sites of melatonin in the cell. The presence of calreticulin was further identified by Western blot analysis, and the lack of endoplasmic reticulum contamination in our material was assessed by Western blot and immunostaining with anti-calnexin Ig. The results suggest that calreticulin may represent a new class of high-affinity melatonin binding sites involved in some functions of the indoleamine including genomic regulation.

Amino Acid Sequence↗

PubMed2002

Inhibition of nNOS activity in rat brain by synthetic kynurenines: structure-activity dependence.

The overstimulation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors is involved in excitotoxicity, a process participating in neurodegeneration that characterizes some neurological disorders and acute cerebral insults. In looking for compounds with neuroprotective properties, a series of kynurenine derivatives were synthesized, and their effects on both the NMDA and nNOS activity in rat striatum were evaluated. Two compounds, 15a (2-acetamido-4-(2-amino-5-methoxyphenyl)-4-oxobutyric acid) and 15c (2-butyramido-4-(2-amino-5-methoxyphenyl)-4-oxobutyric acid), displayed more potent activities than the other synthetic compounds tested for the inhibition of NMDA excitability and nNOS activity. Two other compounds, 18a (2-acetamido-4-(3-methoxyphenyl)-4-oxobutyric acid) and 18c (2-butyramido-4-(3-methoxyphenyl)-4-oxobutyric acid), that have the same structure as 15a and 15c, except the amino group in R(1), showed different effects. Whereas compound 18a showed lower electrophysiological potency than compounds 15a and 15c in the inhibition of the NMDA-dependent excitability, compound 18c showed the opposite effect. Moreover, compounds 18a and 18c were unable to modify nNOS activity. The remaining kynurenines tested behave like compound 18a. These results suggest that a structure-related activity of these synthetic kynurenines and a N-H bond in a specific direction is necessary for some kynurenine analogues to inhibit nNOS activity.

Animals↗

PubMed2002

Melatonin increases the activity of the oxidative phosphorylation enzymes and the production of ATP in rat brain and liver mitochondria.

We recently showed that melatonin counteracted mitochondrial oxidative stress and increased the activity of the mitochondrial oxidative phosphorylation (OXPHOS) enzymes both in vivo and in vitro. To further clarify these effects, we studied here the activity of OXPHOS enzymes and the synthesis of ATP in rat liver and brain mitochondria in vitro. In sub-mitochondrial particles, melatonin increases the activity of the complexes I and IV dose-dependently, the effect being significant between 1 and 10nM. Blue native-PAGE followed by histochemical analysis of the OXPHOS enzymes further showed the melatonin-induced increase of complex I activity. Titration studies show that melatonin counteracts the partial inhibition of complex IV induced by 5 microM potassium cyanide. However, melatonin (up to 5mM) was unable to recover the activity of complex IV when it was completely blocked by 100 microM cyanide. These data suggest that the indoleamine could stimulate the activity of the non-inhibited part of the complex IV. Melatonin also increases the production of ATP in control mitochondria and counteracts the cyanide-induced inhibition of ATP synthesis. These results provide new hormonal mechanism regulating mitochondrial homeostasis and may explain, at least in part, the anti-aging and neuroprotective properties of melatonin.

Adenosine Triphosphate↗

PubMed2002

Evaluation of plasma levels of melatonin after midazolam or sodium thiopental anesthesia in children.

Midazolam and sodium thiopental are two commonly used drugs in anesthesia for minor surgical procedures in children. A relationship exists between benzodiazepines (BNZ), barbiturates and melatonin. Whereas these drugs increase pineal melatonin production, the indoleamine amplifies the effects of both BNZ and barbiturates on the central nervous system (CNS). Our purpose was thus to analyze the plasma levels of melatonin before and during midazolam or sodium thiopental anesthesia in children subjected to ambulatory surgical procedures. Midazolam (0.4 mg/kg) or sodium thiopental (5 mg/kg) were administered i.v. to 33 and 32 children (aged between 2 and 14 yr), respectively, and blood samples were taken before and 5, 10 and 20 min after the drugs were administered. Melatonin was measured in plasma by a commercial radioimmunoassay kit previously standardized in our laboratory. The results showed that neither midazolam nor sodium thiopental anesthesia significantly affected the levels of melatonin studied at anytime. Significant correlations were found comparing the levels of melatonin between the different times studied. These results suggest that midazolam or sodium thiopental did not affect melatonin production by the pineal gland, thus avoiding a possible potentiating effect of the indoleamine on the central effects of these drugs during anesthesia. However, the possibility that changes in melatonin had been masked by the antioxidant role of the neurohormone are discussed.

Adolescent↗

PubMed2002

Circadian rhythms of dopamine and dihydroxyphenyl acetic acid in the mouse striatum: effects of pinealectomy and of melatonin treatment.

The existence of dopamine (DA)-melatonin (aMT) relationships is well documented in several brain areas of the mammalian central nervous system such as the retina and hypothalamus or the nigrostriatal system. For instance, aMT tempers 1 methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced nigrostriatal damage in C57BL/6 mice. In this mouse strain however, rhythmic production of aMT and its possible interaction with striatal DA is still unclear. In the present work we investigated circadian variations in pineal production of aMT and striatal DA levels in C57BL/6 mice. Effects of pinealectomy and aMT administration were also assessed. Intact, pinealectomized and pinealectomized + aMT-treated mice and their respective control groups were sacrificed at six different times during the 24-hour period. In control animals, aMT displayed a circadian rhythm with a narrow peak at midnight. The peak of aMT coincided with the nadir of the DA rhythm present in the striatum. Shortly after the decrease of DA levels, an increase in 3,4-dihydroxyphenylacetic acid (DOPAC), the main DA metabolite, was observed. The rhythmic changes of DA and DOPAC levels in the striatum were blunted by pinealectomy, whereas administration of aMT (0.1-10 mg/kg) during 6 days to pinealectomized mice restored the rhythms in a dose-dependent manner. Striatal levels of 3-methoxytyramine and homovanillic acid did not change during the 24-hour cycle. The serotonergic system, assessed by the determination of 5-hydroxytryptamine and 5-hydroxyindole-3-acetic acid concentration in striatum, did not show significant time-dependent changes in control animals and was not affected by pinealectomy or aMT treatment. These data substantiate the existence of a link between pineal function, melatonin secretion and DA circadian rhythm in the mouse striatum.

3,4-Dihydroxyphenylacetic Acid↗

PubMed2002

Melatonin, mitochondrial homeostasis and mitochondrial-related diseases.

The recently described 'hydrogen hypothesis' invokes metabolic symbiosis as the driving force for a symbiotic association between an anaerobic, strictly hydrogen-dependent organism (the host) and an eubacterium (the symbiont) that is able to respire, but which generates molecular hydrogen as an end product of anaerobic metabolism. The resulting proto-eukaryotic cell would have acquired the essentials of eukaryotic energy metabolism, evolving not only aerobic respiration, but also the cost of oxygen consumption, i.e., generation of reactive oxygen species (ROS) and oxidative damage. Mitochondria contain their own genome with a modified genetic code that is highly conserved among mammals. Control of gene expression suggests that transcription of certain mitochondrial genes may be regulated in response to the redox potential of the mitochondrial membrane. Mitochondria are involved in energy production and conservation, and they have an uncoupling mechanism to produce heat instead of ATP. Also, mitochondria are involved in programmed cell death. Increasing evidence suggests the participation of mitochondria in neurodegenerative and neuromuscular diseases involving alterations in both nuclear (nDNA) and mitochondrial (mtDNA) DNA. Melatonin is now known as a powerful antioxidant and increasing experimental evidence shows its beneficial effects against oxidative stress-induced macromolecular damage and diseases, including those in which mitochondrial function is affected. This review summarizes the data and mechanisms of action of melatonin in relation to mitochondrial pathologies.

Animals↗

PubMedDate not supplied

Synergistic effects of melatonin and deprenyl against MPTP-induced mitochondrial damage and DA depletion.

Previous studies showed a synergistic effect of melatonin and deprenyl against dopamine (DA) autoxidation in vitro. Since oxidative stress is implicated in Parkinson's disease (PD), we explored the effects of melatonin plus deprenyl administration in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD in C57/Bl6 mice. Melatonin, but not deprenyl prevents the inhibition of mitochondrial complex I and the oxidative damage in nigrostriatal neurons induced by MPTP. With the dose used deprenyl recovers 50% DA levels and tyrosine hydroxylase activity depressed by the neurotoxin, normalizing locomotor activity of mice. Melatonin, which was unable to counteract MPTP-induced DA depletion and inhibition of tyrosine hydroxylase activity, potentiates the effect of deprenyl on catecholamine turnover and mice ambulatory activity. These results suggest a dissociation of complex I inhibition from DA depletion in this model of Parkinson's disease. The data also support that a combination of melatonin, which improves mitochondrial electron transport chain and reduces oxidative damage, and deprenyl, which promotes the specific function of the rescued neurons, i.e. DA turnover, may be a promising strategy for the treatment of PD.

3,4-Dihydroxyphenylacetic Acid↗