Influence of aging and growth hormone on different members of the NFkB family and IkB expression in the heart from a murine model of senescence-accelerated aging.

Inflammation is related to several pathological processes. The aim of this study was to investigate the protein expression of the different subunits of the nuclear factor Kappa b (NFkBp65, p50, p105, p52, p100) and the protein expressions of IkB beta and alpha in the hearts from a murine model of accelerated aging (SAM model) by Western blot. In addition, the translocation of some isoforms of NFkB from cytosol to nuclei (NFkBp65, p50, p52) and ATP level content was studied. In addition we investigated the effect of the chronic administration of growth hormone (GH) on these age-related parameters. SAMP8 and SAMR1 mice of 2 and 10 months of age were used (n = 30). Animals were divided into five experimental groups: 2 old untreated (SAMP8/SAMR1), 2 young control (SAMP8/SAMR1) and one GH treated-old groups (SAMP8). Age-related changes were found in the studied parameters. We were able to see decreases of ATP level contents and the translocation of the nuclear factor kappa B p50, p52 and p65 from cytosol to nuclei in old SAMP8 mice together with a decrease of IKB proteins. However p100 and p105 did not show differences with aging. No significant changes were recorded in SAMR1 animals. GH treatment showed beneficial effects in old SAMP8 mice inducing an increase in ATP levels and inhibiting the translocation of some NFkB subunits such as p52. Our results supported the relation of NFkB activation with enhanced apoptosis and pro-inflammatory status in old SAMP8 mice and suggested a selective beneficial effect of the GH treatment, which was able to partially reduce the incidence of some deleterious changes in the heart of those mice.

Melatonin in the oral cavity: physiological and pathological implications.

BACKGROUND AND OBJECTIVES: The purpose of this article was to summarize what is known about the function of melatonin in the oral cavity. MATERIAL AND METHODS: Databases were searched for the relevant published literature to 30 November, 2013. The following search items were used in various combinations: melatonin, gingiva, periodontium, inflammation, herpes, alveolar bone, periodontal ligament, dental implants, xerostomia, methacrylate, chlorhexidine, cancer. The literature uncovered is summarized herein. RESULTS: Salivary melatonin levels exhibit a circadian rhythm with highest values at night. Melatonin has both receptor-mediated and receptor-independent actions in cells of the oral cavity. Melatonin is released into the saliva by the acinar cells of the major salivary glands and via the gingival fluid. Functions of melatonin in the oral cavity are likely to relate primarily to its anti-inflammatory and antioxidant activities. These actions may suppress inflammation of the gingiva and periodontium, reduce alveolar bone loss, abrogate herpes lesions, enhance osteointegration of dental implants, limit oral cancer, and suppress disorders that have a free radical component. Sublingual melatonin tablets or oral melatonin sprays and topical melatonin-containing gel, if used on a regular basis, may improve overall oral health and reduce mucosal lesions. CONCLUSION: Collectively, the results indicate that endogenously-produced and exogenously-applied melatonin are beneficial to the oral cavity.

Melatonin synthetic analogs as nitric oxide synthase inhibitors.

Nitric oxide (NO), which is produced by oxidation of L-arginine to L-citrulline in a process catalyzed by different isoforms of nitric oxide synthase (NOS), exhibits diverse roles in several physiological processes, including neurotransmission, blood pressure regulation and immunological defense mechanisms. On the other hand, an overproduction of NO is related with several disorders as Alzheimer's disease, Huntington's disease and the amyotrophic lateral sclerosis. Taking melatonin as a model, our research group has designed and synthesized several families of compounds that act as NOS inhibitors, and their effects on the excitability of N-methyl-D-aspartate (NMDA)-dependent neurons in rat striatum, and on the activity on both nNOS and iNOS were evaluated. Structural comparison between the three most representative families of compounds (kynurenines, kynurenamines and 4,5-dihydro-1H-pyrazole derivatives) allows the establishment of structure-activity relationships for the inhibition of nNOS, and a pharmacophore model that fulfills all of the observed SARs were developed. This model could serve as a template for the design of other potential nNOS inhibitors. The last family of compounds, pyrrole derivatives, shows moderate in vitro NOS inhibition, but some of these compounds show good iNOS/nNOS selectivity. Two of these compounds, 5-(2-aminophenyl)-1H-pyrrole-2-carboxylic acid methylamide and cyclopentylamide, have been tested as regulators of the in vivo nNOS and iNOS activity. Both compounds prevented the increment of the inducible NOS activity in both cytosol (iNOS) and mitochondria (i-mtNOS) observed in a MPTP model of Parkinson's disease.

Melatonin and its agonists in pain modulation and its clinical application.

Melatonin, the hormone of darkness has many physiological functions in the body and also exerts a number of pharmacological effects. Most of these actions of melatonin are mediated through melatonin membrane receptors like MT1/MT2 receptors or through nuclear orphan receptors like RZR/ROR receptors or through calcium binding proteins in the cytosol. The finding that pain perception is circadian in nature has prompted many to suggest that "pain modulation" is one of the most important physiological functions of melatonin. By using a number of animal models of pain perception, it has been found that melatonin exerts antinociceptive and antiallodynic effects. Number of studies has shown that melatonin modulates pain perception by acting through opioid receptors, NMDA receptors and G-protein, and they have been analyzed using specific antagonists like naloxone or NMDA-G protein receptor antagonists. Recently it has been shown that melatonin exerts its antinociceptive effects through MT1 and MT2 melatonergic receptors located in the dorsal region of the spinal cord as well as in various parts of the brain concerned with pain modulation. Evidences for this have been obtained by using common melatonergic receptor antagonist like luzindole or specific MT2 receptor antagonist like 4P-PDOT or K-185. In a few clinical studies undertaken during surgery, melatonin has been shown to have analgesic effects. Melatonin is emerging as a new analgesic drug with a novel mechanism of actions and has the potential to be used as a natural pain killer in inflammatory, neuropathic pain conditions and also during surgical procedures.

Melatonin reduces membrane rigidity and oxidative damage in the brain of SAMP8 mice.

We evaluated the autophagy-lysosomal pathway and membrane fluidity in brain cells and mitochondrial membranes obtained from senescence-accelerated (SAMP(8)) and senescence-resistant (SAMR(1)) mice at 5 and 10 months of age. Moreover, we studied whether chronic treatment from age 1 to 10 months with melatonin stabilizes membrane fluidity. Fluidity was measured by polarization changes of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene-p-toluene sulfonate. Results showed that in untreated animals at 5 months of age, synaptosomal and mitochondrial fluidity was decreased in SAMP(8) compared to SAMR(1), as was the cathepsin D/B ratio, indicating dysfunction of the autophagy-lysosomal pathway. Moreover, we detected synaptosomal rigidity and programmed cell death capability in both groups at 10 months of age. Mitochondrial fluidity, however, did not show a significant age-dependent change but was lower in SAMP(8) than in SAMR(1) at the 5- and 10-month time points. Melatonin administration prevented rigidity in the mitochondrial membrane and seemed to decrease age-related autophagy-lysosomal alterations. These data suggest that melatonin may act to slow down the aging process because of its ability to enhance membrane fluidity and maintain structural pathways.

Melatonin, a potential therapeutic agent for smooth muscle-related pathological conditions and aging.

Increases or decreases in the contractile response of smooth muscle underlie important pathological conditions such as hypertension, incontinence and altered gastrointestinal transit. These disorders are also frequently encountered in the aged population. Oxidative stress and inflammation are key features in the initiation, progression, and clinical manifestations of smooth muscle disorders. Melatonin, the major secretory product of the pineal gland, has free radical scavenging and antioxidative properties and protects against oxidative insult. Recently, widespread interest has grown regarding the apparent protective effects of melatonin on smooth muscle dysfunction. "In vitro" studies have shown that melatonin decreased vascular tone of vascular beds from control, hypertensive or aged animals, through the reduction of adrenergic contraction and the increase in acetylcholine-induced relaxation. "In vivo", melatonin also attenuates sympathetic tone by direct activation of melatonin receptors, scavenging free radicals or increasing NO availability in the central nervous system. In the gastrointestinal tract, melatonin treatment improves age-related impairments in gallbladder contractility and prevents deleterious effects of cholecystitis on smooth muscle and the enteric nervous system through suppression of oxidative stress. In addition, melatonin improves colonic transit time in constipation-predominant IBS patients. Melatonin is also able to restore impaired contractility of the detrusor muscle from old animals through normalization of Ca(2+) dependent and independent contraction, mitochondrial polarity, neuromuscular function and oxidative stress, which would explain the effects of melatonin counteracting cystometric changes in senescent animals. It also reverses bladder damage following ischemia/reperfusion. In conclusion, melatonin may be a promising candidate for future research of agents that modulate smooth muscle motility.

[Melatonin, synthetic analogs, and the sleep/wake rhythm]. / Melatonina, analogos sinteticos y el ritmo sueno/vigilia.

INTRODUCTION: Melatonin, a widespread hormone in the animal kingdom, is produced by several organs and tissues besides the pineal gland. Whilst extrapineal melatonin behaves as a cytoprotective molecule, the pineal produces the hormone in a rhythmic manner. The discovery of melatonin in 1958, and the characterization of its synthesis somewhat later, let to the description of its photoperiodic regulation and its relationship with the biological rhythms such as the sleep/wake rhythm. DEVELOPMENT: The suprachiasmatic nuclei are the anatomical seat of the biological clock, represented by the clock genes, which code for the period and frequency of the rhythms. The photoperiod synchronizes the activity of the auprachiasmatic biological clock, which in turn induces the melatonin's rhythm. The rhythm of melatonin, peaking at 2-3 am, acts as an endogenous synchronizer that translates the environmental photoperiodic signal in chemical information for the cells. The sleep/wake cycle is a typical biological rhythm synchronized by melatonin, and the sleep/wake cycle alterations of chronobiological origin, are very sensitive to melatonin treatment. Taking advantage of the chronobiotic and antidepressive properties of melatonin, a series of synthetic analogs of this hormone, with high interest in insomnia, are now available. CONCLUSIONS: Melatonin is a highly effective chronobiotic in the treatment of chronobiological alterations of the sleep/wake cycle. From a pharmacokinetic point of view, the synthetic drugs derived from melatonin are interesting tools in the therapy of these alterations.

Melatonin protects the mitochondria from oxidative damage reducing oxygen consumption, membrane potential, and superoxide anion production.

The role of melatonin in improving mitochondrial respiratory chain activity and increasing ATP production in different experimental conditions has been widely reported. To date, however, the mechanism(s) involved are largely unknown. Using high-resolution respirometry, fluorometry and spectrophotometry we studied the effects of melatonin on normal mitochondrial functions. Mitochondria were recovered from mouse liver cells and incubated in vitro with melatonin at concentrations ranging from 1 nm to 1 mm. Melatonin decreased oxygen consumption concomitantly with its concentration, inhibited any increase in oxygen flux in the presence of an excess of ADP, reduced the membrane potential, and consequently inhibited the production of superoxide anion and hydrogen peroxide. At the same time it maintained the efficiency of oxidative phosphorylation and ATP synthesis while increasing the activity of the respiratory complexes (mainly complexes I, III, and IV). The effects of melatonin appeared to be due to its presence within the mitochondria, since kinetic experiments clearly showed its incorporation into these organelles. Our results support the hypothesis that melatonin, together with hormones such as triiodothyronine, participates in the physiological regulation of mitochondrial homeostasis.

Melatonin increases following convulsive seizures may be related to its anticonvulsant properties at physiological concentrations.

Melatonin ( N-acetyl-5-methoxytryptamine, aMT) is an indoleamine produced by several organs and tissues including the pineal gland. Melatonin (aMT) modulates the activity of the brain, mainly acting on both GABA and glutamate receptors. Previous studies have shown the participation of melatonin in the control of convulsive crises, suggesting that aMT concentration increases during seizures, and that patients with seizures of diverse origins show an alteration of the aMT rhythm. However, what is not known is the duration of the aMT response to seizures, and whether aMT changes during seizures could be a marker of the disease. For this reason, the serum levels of aMT in 54 children with a convulsive crisis, febrile and epileptic, were analyzed during the crisis, as well as at 1 h and 24 hours after the seizure. The results show that aMT significantly increases during the seizure (Day group, 75.64+/-45.91 and Night group, 90.69+/-51.85 pg/mL), with normal values being recovered 1 h later (Day group, 26.33+/-10.15 and Night group, 27.78+/-7.82 pg/mL) and maintained for up to 24 hours, when the circadian variation of aMT returns to the normal acrophase. Due to the interindividual variation of aMT levels among healthy people, a single determination of the indoleamine concentration is not a suitable marker of the existence of a convulsive crisis unless the circadian profile of aMT secretion in the patient is known. The results obtained also support the view that the stimulation of aMT production by the convulsive crisis may participate in the response of the organism against the seizures.

Melatonin expression in periodontal disease.

BACKGROUND AND OBJECTIVE: It was the purpose of this study to examine the relationship between periodontal diseases and melatonin level. MATERIAL AND METHODS: Forty-six patients with periodontal disease, together with 26 age- and gender-matched healthy controls, were included. Periodontal status was assessed using the Community Periodontal Index. Plasma and salivary melatonin levels were determined using specific commercial radioimmunoassays, whereas lymphocyte subpopulations (e.g. CD3, CD4, CD8, C19 and natural killer cells) were analyzed using flow cytometry. RESULTS: Patients with periodontal disease had significantly (p < 0.001) lower plasma (9.46 +/- 3.18 pg/mL) and saliva (2.55 +/- 0.99 pg/mL) melatonin levels than healthy control patients (14.33 +/- 4.05 and 4.22 +/- 0.87 pg/mL, respectively). A biphasic relationship was observed between plasma melatonin levels and Community Periodontal Indices. The plasma melatonin level was reduced in patients with a lower Community Periodontal Index value (1 or 2) and increased in patients with a higher Community Periodontal Index value (3 or 4). Salivary melatonin parallels the changes of plasma melatonin. The higher the Community Periodontal Index, the older the patient and the higher the total lymphocyte counts. CD4 concentrations also increased as the disease worsened. CONCLUSION: The results obtained from this study suggest that melatonin could act as a protective function in fighting periodontal infection. However, further studies in this area are encouraged.

Melatonin administration prevents cardiac and diaphragmatic mitochondrial oxidative damage in senescence-accelerated mice.

Cardiac and diaphragmatic mitochondria from male SAMP8 (senescent) and SAMR1 (resistant) mice of 5 or 10 months of age were studied. Levels of lipid peroxidation (LPO), glutathione (GSH), GSH disulfide (GSSG), and GSH peroxidase and GSH reductase (GRd) activities were measured. In addition, the effect of chronic treatment with the antioxidant melatonin from 1 to 10 months of age was evaluated. Cardiac and diaphragmatic mitochondria show an age-dependent increase in LPO levels and a reduction in GSH:GSSG ratios. Chronic treatment with melatonin counteracted the age-dependent LPO increase and GSH:GSSG ratio reduction in these mitochondria. Melatonin also increased GRd activity, an effect that may account for the maintenance of the mitochondrial GSH pool. Total mitochondrial content of GSH increased after melatonin treatment. In general, the effects of age and melatonin treatment were similar in senescence-resistant mice (SAMR1) and SAMP8 cardiac and diaphragmatic mitochondria, suggesting that these mice strains display similar mitochondrial oxidative damage at the age of 10 months. The results also support the efficacy of long-term melatonin treatment in preventing the age-dependent mitochondrial oxidative stress.

Binding properties and immunolocalization of a fatty acid-binding protein in Giardia lamblia.

We describe here a fatty acid-binding protein (FABP) isolated and purified from the parasitic protozoon Giardia lamblia. The protein has a molecular mass of 8 kDa and an isoelectric point of 4.96. A Scatchard analysis of the data at equilibrium revealed a dissociation constant of 3.12 x 10(-8) M when the labeled oleic acid was displaced by a 10-fold greater concentration of unlabeled oleic acid. Testosterone, sodium desoxycholate, taurocholate, metronidazol, and alpha-tocopherol, together with butyric, arachidonic, palmitic, retinoic, and glycocholic acids, were also bound to the protein. Assays with polyclonal antibodies revealed that the protein is located in the ventral disk and also appears in the dorsal membrane, the cytoplasm, and in the vicinity of the lipid vacuoles.

Mechanisms of N-methyl-D-aspartate receptor inhibition by melatonin in the rat striatum.

N-methyl-D-aspartate (NMDA) receptor activation comprises multiple regulatory sites controlling Ca2+ influx into the cell. NMDA-induced increases in intracellular [Ca(+2)] lead to nitric oxide (NO) production through activation of neuronal NO synthase (nNOS). Melatonin inhibits either glutamate or NMDA-induced excitation, but the mechanism of this inhibition is unknown. In the present study, the mechanism of melatonin action in the rat striatum was studied using extracellular single unit recording of NMDA-dependent neuronal activity with micro-iontophoresis. Melatonin inhibited neuronal excitation produced by either NMDA or L-arginine. The effects of both NMDA and L-arginine were blocked by nitro-L-arginine methyl ester, suggesting that nNOS participates in responses to NMDA. However, excitation of NMDA-sensitive neurones induced by the NO donor sodium nitroprusside was only slightly modified by melatonin. Melatonin iontophoresis also counteracted excitation induced by tris(2-carboxyethyl)phosphine hydrochloride, showing that the redox site of the NMDA receptor may be a target for melatonin action. The lack of effects of the membrane melatonin receptor ligands luzindole, 4-phenyl-2-propionamidotetralin and 5-methoxycarbonylamino-N-acetyltryptamine, and the nuclear melatonin ligand, CGP 52608, a thiazolidine dione, excluded the participation of known membrane and nuclear receptors for melatonin. The data suggest that inhibition of NMDA-dependent excitation by melatonin involves both nNOS inhibition and redox site modulation.

Melatonin improves deferoxamine antioxidant activity in protecting against lipid peroxidation caused by hydrogen peroxide in rat brain homogenates.

Deferoxamine (DF) is an antioxidant molecule because of its ability to chelate iron. This study compared the ability of DF alone or in combination with melatonin, 5-methoxytryptophol or pinoline in preventing lipid peroxidation due to hydrogen peroxide (H(2)O(2)) in rat brain homogenates. Malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA) in the homogenates were measured as indices of lipid peroxidation. Incubation of homogenates with DF reduced, in a dose-dependent manner, MDA+4-HDA formation due to H(2)O(2). When melatonin, 5-methoxytryptophol or pinoline were added to the incubation medium, the efficacy of DF in preventing lipid peroxidation was enhanced. These cooperative effects between DF, melatonin, and related pineal products may be important in protecting tissues from the oxidative stress due to iron overload.

Melatonin and beta-endorphin changes in children sensitized to olive and grass pollen after treatment with specific immunotherapy.

BACKGROUND: Specific immunotherapy for respiratory allergy, a seasonal disease, significantly reduces the inflammatory process, attenuating the clinical symptoms. The mechanism for the clinical beneficial effect of immunotherapy has not yet been clarified. Melatonin shows a circadian and seasonal variation and together with the endogenous opioid system plays an immunomodulatory role acting on both specific and nonspecific immunity responses. Thus, the possibility that immunotherapy involved changes in the melatonin-opioid system was investigated. METHODS: Thirty-five children aged 3-15 years with rhinitis and asthma due to olive + grass pollen sensitization were studied. The patients were treated with depot extracts containing the identified allergens with increasing doses from 1 to 1,000 IU/ml during 3 months. Melatonin, beta-endorphin, total and specific IgE and IgG4 were determined before and after treatment. RESULTS: All children showed a significant improvement of their symptoms at the end of the treatment, coinciding with a significant drop of both melatonin and beta-endorphin levels. Total IgE decreased in most of the cases although the mean values did not show significant changes. Specific IgE and IgG4 were also unchanged. A significant correlation between melatonin and beta-endorphin and between beta-endorphin and IgG4 was found before immunotherapy, and these correlations disappeared when the treatment was finished. CONCLUSIONS: The decrease in the levels of melatonin and beta-endorphin suggests the disappearance of their stimulating influence on the immune system. Thus, hyposensitization after immunotherapy may involve, at least in part, the inhibition of the immunoenhancing properties of the melatonin-opioid system.

N-acetylserotonin suppresses hepatic microsomal membrane rigidity associated with lipid peroxidation.

N-acetylserotonin, the immediate precursor of melatonin in the tryptophan metabolic pathway in the pineal gland, has been reported to be an antioxidant. The aim of this work was to test the effect of N-acetylserotonin in stabilizing biological membranes against oxidative stress. Hepatic microsomal membranes from male adult rats were incubated with N-acetylserotonin (0.001-3 mM) before inducing lipid peroxidation using FeCl(3), ADP and NADPH. Control experiments were done by incubating microsomal membranes with N-acetylserotonin in the absence of lipid peroxidation-inducing drugs. Membrane fluidity was assessed by fluorescence spectroscopy and malonaldehyde plus 4-hydroxyalkenals concentrations were measured to estimate the degree of lipid peroxidation. Free radicals induced by the combination of FeCl(3)+ADP+NADPH produced a significant decrease in the microsomal membrane fluidity, which was associated with an increase in the malonaldehyde plus 4-hydroxyalkenals levels. These changes were suppressed in a concentration-dependent manner when N-acetylserotonin was added in the incubation buffer. In the absence of lipid peroxidation, N-acetylserotonin (0.001-3 mM) did not change membrane fluidity nor malonaldehyde plus 4-hydroxyalkenals levels. These results suggest that the protective role of N-acetylserotonin in preserving optimal levels of fluidity of the biological membranes may be related to its ability to reduce lipid peroxidation.

Free radical-mediated molecular damage. Mechanisms for the protective actions of melatonin in the central nervous system.

This review briefly summarizes the multiple actions by which melatonin reduces the damaging effects of free radicals and reactive oxygen and nitrogen species. It is well documented that melatonin protects macromolecules from oxidative damage in all subcellular compartments. This is consistent with the protection by melatonin of lipids and proteins, as well as both nuclear and mitochondrial DNA. Melatonin achieves this widespread protection by means of its ubiquitous actions as a direct free radical scavenger and an indirect antioxidant. Thus, melatonin directly scavenges a variety of free radicals and reactive species including the hydroxyl radical, hydrogen peroxide, singlet oxygen, nitric oxide, peroxynitrite anion, and peroxynitrous acid. Furthermore, melatonin stimulates a number of antioxidative enzymes including superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase. Additionally, melatonin experimentally enhances intracellular glutathione (another important antioxidant) levels by stimulating the rate-limiting enzyme in its synthesis, gamma-glutamylcysteine synthase. Melatonin also inhibits the proxidative enzymes nitric oxide synthase and lipoxygenase. Finally, there is evidence that melatonin stabilizes cellular membranes, thereby probably helping them resist oxidative damage. Most recently, melatonin has been shown to increase the efficiency of the electron transport chain and, as a consequence, to reduce election leakage and the generation of free radicals. These multiple actions make melatonin a potentially useful agent in the treatment of neurological disorders that have oxidative damage as part of their etiological basis.