Attenuation of cardiac mitochondrial dysfunction by melatonin in septic mice

The existence of an inducible mitochondrial nitric oxide synthase has been recently related to the nitrosative/oxidative damage and mitochondrial dysfunction that occurs during endotoxemia. Melatonin inhibits both inducible nitric oxide synthase and inducible mitochondrial nitric oxide synthase activities, a finding related to the antiseptic properties of the indoleamine. Hence, we examined the changes in inducible nitric oxide synthase/inducible mitochondrial nitric oxide synthase expression and activity, bioenergetics and oxidative stress in heart mitochondria following cecal ligation and puncture-induced sepsis in wild-type (iNOS(+/+)) and inducible nitric oxide synthase-deficient (iNOS(-/-)) mice. We also evaluated whether melatonin reduces the expression of inducible nitric oxide synthase/inducible mitochondrial nitric oxide synthase, and whether this inhibition improves mitochondrial function in this experimental paradigm. The results show that cecal ligation and puncture induced an increase of inducible mitochondrial nitric oxide synthase in iNOS(+/+) mice that was accompanied by oxidative stress, respiratory chain impairment, and reduced ATP production, although the ATPase activity remained unchanged. Real-time PCR analysis showed that induction of inducible nitric oxide synthase during sepsis was related to the increase of inducible mitochondrial nitric oxide synthase activity, as both inducible nitric oxide synthase and inducible mitochondrial nitric oxide synthase were absent in iNOS(-/-) mice. The induction of inducible mitochondrial nitric oxide synthase was associated with mitochondrial dysfunction, because heart mitochondria from iNOS(-/-) mice were unaffected during sepsis. Melatonin treatment blunted sepsis-induced inducible nitric oxide synthase/inducible mitochondrial nitric oxide synthase isoforms, prevented the impairment of mitochondrial homeostasis under sepsis, and restored ATP production. These properties of melatonin should be considered in clinical sepsis.     

Melatonin effects on gut motility are independent of the relaxation mediated by the nitrergic system in the goldfish

Melatonin is a key neuroendocrine transducer in the circadian organization of vertebrates. However, its role in gastrointestinal physiology has not been explored in depth. In goldfish, a role for melatonin as a modulator of intestinal motility has been reported, whereby it attenuates the cholinergic contraction. The aim of the present work was to investigate this relaxation induced by melatonin in the gut smooth muscle of the goldfish, studying the possible involvement of nitric oxide. An in vitro model of isolated goldfish intestine was used to test the effects on intestinal motility. The addition of melatonin (10 pM-100 μM) to the organ bath relaxed acetylcholine- and serotonin-stimulated gut strips, but no effect was observed on KCl-contracted preparations. The addition of L-NAME (nitric oxide synthase inhibitor) increased the amplitude of the spontaneous slow waves, while sodium nitroprusside (SNP, nitric oxide donor) abolished them. All these results support a role for the nitrergic system in goldfish gut motility. However, neither L-NAME, nor SNP nor the nitric oxide precursor, l-arginine, modified the melatonin relaxing effect. These results highlight the existence of a basal nitrergic tone in the gut of goldfish, where melatonin would exert a calcium-dependent, nitric oxide-independent relaxing effect on serotonergic and cholinergic contraction.     

Melatonin: New Places in Therapy

The fact that the full extent of the function of the pineal gland has not yet been elucidated, has stimulated melatonin research worldwide. This review introduces melatonin’s mechanism of action, direct and indirect antioxidant actions as well as the antioxidant properties of its metabolites, 6-hydroxymelatonin (6-OHM) and N-acetyl-N-formyl-5-methoxykynurenamine (AFMK). At present the mechanism of action is proposed to be receptor-, protein- and nonprotein-mediated. From its popular role in the treatment of jetlag, melatonin is now implicated in the reduction of oxidative stess, both as a free radical scavenger and antioxidant. Melatonin’s direct scavenging action in respect of the following will be discussed: superoxide anions, hydrogen peroxide, hydroxyl radicals, singlet oxygen, peroxy radicals and nitric oxide/peroxy nitrite anions. In addition melatonin also possesses indirect antioxidant activity and the role of its metabolites, AFMK and 6-OHM will be presented. It is these free radical scavenging and antioxidant properties of melatonin that has shifted the focus from that of merely strengthening circadian rhythms to that of neuroprotectant: a new place in therapy.     

Mitochondria: The hands of the clock?

The output of the master clock to control peripheral circadian functions and temporal organization in metazoans is unknown. We recently reported a periodic dip of lipidperoxidation (PDL) at 03 h in humans by monitoring the serum levels of malondialdehyde (MDA), a marker of lipidperoxidation, over a 24-hour period and suggested that PDL may represent a redox signal of the master clock. Mitochondria are considered to be the major source of lipidperoxidation in most tissues. Their activity is known to be disrupted in many malignant tissues. To test the possible role of mitochondrial activity on the induction of the PDL, we monitored the daily MDA levels in the serum of 16 cancer patients, suffering from different kinds of malignant tumors, and compared them with those found in the healthy humans of our previous study. Discriminant analysis revealed a significant difference between both groups only at 03 h ( p = 0.000001), thus indicating the critical relevance of this time point for differences between both groups. In the light of the emerging role of the mitochondrion in cell signaling, we suggest that the signal transduction from the master clock may be related to central and peripheral mitochondrial activity and its ROS (reactive oxygen species) generation. This notion could involve nitric oxide, as a known potent regulator of mitochondrial function and as an essential part of the mitochondrial signaling pathway. The PDL could additionally be useful as a circadian phase marker in chronobiology research.     

Melatonin modulates the effects of gastric injury in rats: role of prostaglandins and nitric oxide

Experimental evidence has been presented connecting melatonin with the prevention or treatment of gastrointestinal disorders either by the scavenging properties of active oxygen or by receptor-mediated stimulation of gene expression of neutralizing enzymes. Prostaglandins and nitric oxide are important neuroimmunomodulators in digestive physiology and different studies have indicated that the protective properties of melatonin may be explained by prostaglandin and/or nitric oxide mechanisms. The aim of the present study was to examine the effect of intraperitoneal administration of melatonin on in vivo changes in PGE(2), generated in gastric mucosal lesions by ischemia-reperfusion. Cyclic GMP nucleotide was also studied as an index of the principal enzymatic activity involved in the metabolism of nitric oxide, the nitric oxide synthase. The different immunological tests showed that the intraperitoneal administration of melatonin prevents the postischemic decrease in prostaglandins. The concentration of this eicosanoid in the rat mucosa treated with 20 mg.kg(-1) of melatonin was significantly higher (p < 0.05) than that in the control rats. The amount of cyclic GMP in the stomach decreased because of ischemia-reperfusion. In treated animals however, a marked increase occurred in concentrations of GMP, but the difference was not statistically significant. The results suggest that the mechanism of protection afforded by melatonin against lesions induced by gastric ischemia-reperfusion may be due to stimulation of the synthesis of eicosanoid protectors during the ischemic process.     

The protective role of endogenous melatonin in carrageenan-induced pleurisy in the rat.

Peroxynitrite, a potent cytotoxic oxidant formed by the reaction of nitric oxide (NO) with the superoxide anion, was recently proposed to play a major pathogenic role in the inflammatory process. Here we have investigated the effects of endogenous melatonin, a known scavenger of peroxynitrite, in rats subjected to carrageenan-induced pleurisy. Endogenous melatonin was depleted in rats maintained on 24 h light cycle for 1 wk. In vivo depletion of endogenous melatonin enhanced the carrageenan-induced degree of pleural exudation and polymorphonuclear leukocyte migration in rats subjected to carrageenan-induced pleurisy. Lung myeloperoxidase activity and lipid peroxidation were significantly increased in melatonin-deprived rats. However, the inducible NO synthase in lung samples was unaffected by melatonin depletion. Immunohistochemical analysis for nitrotyrosine revealed a positive staining in lungs from carrageenan-treated rats that was markedly enhanced in melatonin-deprived rats. Furthermore, melatonin depletion significantly increased peroxynitrite formation as measured by the oxidation of the fluorescent dye dihydrorhodamine 123, enhanced DNA damage and the decrease in mitochondrial respiration and reduced the cellular levels of NAD+ in macrophages harvested from the pleural cavity of rats subjected to carrageenan-induced pleurisy. In vivo treatment with exogenous melatonin (15 mg/kg intraperitoneal) significantly reversed the effects of melatonin depletion. Thus, endogenous melatonin plays an important protective role against carrageenan-induced local inflammation.     

Circadian regulation of bird song, call, and locomotor behavior by pineal melatonin in the zebra finch

As both a photoreceptor and pacemaker in the avian circadian clock system, the pineal gland is crucial for maintaining and synchronizing overt circadian rhythms in processes such as locomotor activity and body temperature through its circadian secretion of the pineal hormone melatonin. In addition to receptor presence in circadian and visual system structures, high-affinity melatonin binding and receptor mRNA are present in the song control system of male oscine passeriform birds. The present study explores the role of pineal melatonin in circadian organization of singing and calling behavior in comparison to locomotor activity under different lighting conditions. Similar to locomotor activity, both singing and calling behavior were regulated on a circadian basis by the central clock system through pineal melatonin, since these behaviors free-ran with a circadian period and since pinealectomy abolished them in constant environmental conditions. Further, rhythmic melatonin administration restored their rhythmicity. However, the rates by which these behaviors became arrhythmic and the rates of their entrainment to rhythmic melatonin administration differed among locomotor activity, singing and calling under constant dim light and constant bright light. Overall, the study demonstrates a role for pineal melatonin in regulating circadian oscillations of avian vocalizations in addition to locomotor activity. It is suggested that these behaviors might be controlled by separable circadian clockworks and that pineal melatonin entrains them all through a circadian clock.     

Melatonin Prevents Cyclosporine-induced Nephrotoxicity in Isolated and Perfused Rat Kidney

Cyclosporine A (CsA) is a potent and effective immunosuppressive agent, but its action is frequently accompanied by severe renal toxicity. The precise mechanism by which CsA causes renal injury is not known. Reactive oxygen species (ROS) have been shown to play a role, since CsA-induced renal lipid peroxidation is attenuated in vivo and in vitro by the concomitant administration of antioxidants such as vitamin E. We show here the effect of the antioxidant melatonin (MLT), a hormone produced by the pineal gland during the dark phase of the circadian cycle, in a model of CsA nephrotoxicity in the isolated and perfused rat kidney. Kidneys isolated from rats were divided into seven groups. At the end of perfusion, malondialdehyde and 4-hydroxyalkenals (MDA+4-HDA), metabolites of nitric oxide N O 2 &#109 +N O 3 &#109 were measured and histopathological examination was performed. CsA treatment induced a significant increase in MDA+4-HDA while not affecting the nitric oxide metabolite level. MLT remarkably prevented glomerular collapse and tubular damage as revealed by morphometric analysis. Our study suggests that lipid peroxidation is an early important event in the pathogenesis of CsA nephrotoxicity and that MLT is able to protect kidneys from CsA at a relatively low concentration.     

Melatonin prevents cyclosporine-induced nephrotoxicity in isolated and perfused rat kidney

Cyclosporine A (CsA) is a potent and effective immunosuppressive agent, but its action is frequently accompanied by severe renal toxicity. The precise mechanism by which CsA causes renal injury is not known. Reactive oxygen species (ROS) have been shown to play a role, since CsA-induced renal lipid peroxidation is attenuated in vivo and in vitro by the concomitant administration of antioxidants such as vitamin E. We show here the effect of the antioxidant melatonin (MLT), a hormone produced by the pineal gland during the dark phase of the circadian cycle, in a model of CsA nephrotoxicity in the isolated and perfused rat kidney. Kidneys isolated from rats were divided into seven groups. At the end of perfusion, malondialdehyde and 4-hydroxyalkenals (MDA+4-HDA), metabolites of nitric oxide N O 2 - +N O 3 - were measured and histopathological examination was performed. CsA treatment induced a significant increase in MDA+4-HDA while not affecting the nitric oxide metabolite level. MLT remarkably prevented glomerular collapse and tubular damage as revealed by morphometric analysis. Our study suggests that lipid peroxidation is an early important event in the pathogenesis of CsA nephrotoxicity and that MLT is able to protect kidneys from CsA at a relatively low concentration.     

Entrainment of the circadian locomotor activity rhythm in the Japanese newt by melatonin injections

We examined whether melatonin can act as a synchronizing agent within the circadian system of amphibians by testing the ability of melatonin injections to entrain the circadian locomotor activity rhythm of a newt (Cynops pyrrhogaster). Under constant darkness, all newts (13 cases) showing the free-running rhythms were subcutaneously injected with 10 g melatonin at the same time every other day for at least 30 days. Subsequently, they were injected with vehicle (1% ethanolic saline) instead of melatonin for at least another 30 days. In 10 of the 13 newts, the locomotor activity rhythms could be entrained to a period of 24 h by melatonin injections but not by vehicle injections. During the entrained steady-state, the active phase of an activity-rest cycle preceded the time of melatonin injections as previously reported in other diurnal species. These results suggest that the endogenous circadian rhythm of melatonin concentration may be involved in synchronizing circadian oscillator(s) within the newt's circadian system.     

Feeding melatonin enhances the phase shifting response to triazolam in both young and old golden hamsters

Aging alters many aspects of circadian rhythmicity, including responsivity to phase-shifting stimuli and the amplitude of the rhythm of melatonin secretion. As melatonin is both an output from and an input to the circadian clock, we hypothesized that the decreased melatonin levels exhibited by old hamsters may adversely impact the circadian system as a whole. We enhanced the diurnal rhythm of melatonin by feeding melatonin to young and old hamsters. Animals of both age groups on the melatonin diet showed larger phase shifts than control-fed animals in response to an injection with the benzodiazepine triazolam at a circadian time known to induce phase advances in the activity rhythm of young animals. Thus melatonin treatment can increase the sensitivity of the circadian timing system of young animals to a nonphotic stimulus, and the ability to increase this sensitivity persists into old age, indicating exogenous melatonin might be useful in reversing at least some age-related changes in circadian clock function.     

<Emphasis Type="Italic">In Vitro</Emphasis>, Melatonin Treatment Decreases Nitric Oxide Levels in Murine Splenocytes Cultured with the Venezuelan Equine Encephalomyelitis Virus

The purpose of this work was to determine the effect of melatonin on the nitric oxide levels in murine splenocytes cultured with the Venezuelan equine encephalomyelitis virus. After incubation, nitric oxide levels were measured by the diazotization assay. Those cultures with the Venezuelan equine encephalomyelitis virus increased nitric oxide levels. Splenocytes infected and treated with 100 and 150 μg/ml of melatonin, decreased significantly the nitric oxide levels when compared to infected and non-treated splenocytes. These findings show that splenocytes infected with the Venezuelan equine encephalomyelitis virus generate important amounts of nitric oxide and suggest that melatonin protects the mice infected with the Venezuelan equine encephalomyelitis virus by a mechanism involving the decreasing of nitric oxide concentrations in tissue.     

Identification of an inducible nitric oxide synthase in diaphragm mitochondria from septic mice: its relation with mitochondrial dysfunction and prevention by melatonin

Sepsis provokes an induction of inducible nitric oxide synthase (iNOS) and melatonin down-regulates its expression and activity. Looking for an inducible mtNOS isoform, we induced sepsis by cecal ligation and puncture in both normal and iNOS knockout mice and studied the changes in mtNOS activity. We also studied the effects of mtNOS induction in mitochondrial function, and the role of melatonin against induced mtNOS and mitochondrial dysfunction. The activity of mtNOS and nitrite levels significantly increased after sepsis in iNOS+/+ mice. These animals showed a significant inhibition of the respiratory chain activity and an increase in mitochondrial oxidative stress, reflected in the disulfide/glutathione ratio, glutathione redox cycling enzymes activity and lipid peroxidation levels. Interestingly, mtNOS activity remained unchanged in iNOS-/- septic mice, and mitochondria of these animals were unaffected by sepsis. Melatonin administration to iNOS+/+ mice counteracted mtNOS induction and respiratory chain failure, restoring the redox status. The results support the existence of an inducible mtNOS that is likely coded by the same gene as iNOS. The results also suggest that sepsis-induced mtNOS is responsible for the increase of mitochondrial impairment due to oxidative stress in sepsis, perhaps due to the high production of NO. Melatonin treatment prevents mitochondrial failure at the same extend as the lack of iNOS gene.     

X-ray irradiation stimulates the mitochondrial-dependent generation of reactive oxygen and nitrogen species in K562 leukemia cells

The dynamics of radiation-induced oxidative and nitrative stress, the source of oxygen and nitrogen reactive species in cancer cell line K562 and the role of mitochondria in these processes have been studied. The study was performed using K562 leukemia cell cultures. Intracellular concentration of reactive oxygen species (ROS), nitrogen oxide, and the mitochondrial potential were analyzed after 15, 30 min, 1, 4, 8, 12, 24, and 48 h after irradiation by X-rays at a dose of 4 and 12 Gy. Radiation-induced generation of ROS in K562 cells has two time peaks, the first peak was recorded after 30 min and the second 24 h after exposure to X-rays. Mitochondria are responsible for the increase of the ROS concentration in the period of 12-48 h after irradiation. The increase in ROS concentrations is accompanied by the increase of the mitochondrial potential. The intracellular concentration of nitric oxide begins to grow 8 h after exposure. The increase in the mitochondria-dependent ROS production is accompanied by the increase in the intracellular concentration of nitric oxide.     

Circadian Rhythms of Oxidative Phosphorylation: Effects of Rotenone and Melatonin on Isolated Rat Brain Mitochondria

Mitochondrial experiments are of increasing interest in different fields of research. Inhibition of mitochondrian activities seems to play a role in Parkinson's disease and in this regard several animal models have used inhibitors of mitochondrial respiration such as rotenone or MPTP. Most of these experiments were done during the daytime. However, there is no reason for mitochondrial respiration to be constant during the 24h. This study investigated the circadian variation of oxidative phosphorylation in isolated rat brain mitochondria and the administration-time-dependent effect of rotenone and melatonin. The respiratory control ratio, state 3 and state 4, displayed a circadian fluctuation. The highest respiratory control ratio value (3.01) occurred at 04:00h, and the lowest value (2.63) at 08:00h. The highest value of state 3 and state 4 oxidative respiration occurred at 12:00h and the lowest one at 20:00h. The 24h mean decrease in the respiratory control ratio following incubation with melatonin and rotenone was 7 and 32%, respectively; however, the exact amount of the inhibition exerted by these agents varied according to the time of the mitochondria isolation. Our results show the time of mitochondrial isolation could lead to interindividual variability. When studies require mitochondrial isolation from several animals, the time between animal experiments has to be minimized. In oxidative phosphorylation studies, the time of mitochondria isolation must be taken into account, or at least specified in the methods section.     

Circadian rhythms of oxidative phosphorylation: effects of rotenone and melatonin on isolated rat brain mitochondria

Mitochondrial experiments are of increasing interest in different fields of research. Inhibition of mitochondrian activities seems to play a role in Parkinson's disease and in this regard several animal models have used inhibitors of mitochondrial respiration such as rotenone or MPTP. Most of these experiments were done during the daytime. However, there is no reason for mitochondrial respiration to be constant during the 24h. This study investigated the circadian variation of oxidative phosphorylation in isolated rat brain mitochondria and the administration-time-dependent effect of rotenone and melatonin. The respiratory control ratio, state 3 and state 4, displayed a circadian fluctuation. The highest respiratory control ratio value (3.01) occurred at 04:00h, and the lowest value (2.63) at 08:00h. The highest value of state 3 and state 4 oxidative respiration occurred at 12:00h and the lowest one at 20:00h. The 24h mean decrease in the respiratory control ratio following incubation with melatonin and rotenone was 7 and 32%, respectively; however, the exact amount of the inhibition exerted by these agents varied according to the time of the mitochondria isolation. Our results show the time of mitochondrial isolation could lead to interindividual variability. When studies require mitochondrial isolation from several animals, the time between animal experiments has to be minimized. In oxidative phosphorylation studies, the time of mitochondria isolation must be taken into account, or at least specified in the methods section.     

Physiological concentrations of melatonin inhibit nitric oxide synthase in rat cerebellum

In the present paper we show the inhibitory effect of melatonin on rat cerebellar nitric oxide synthase (NOS) activity. NO production was monitored by the stoichiometric conversion of L-arginine to L-citrulline. The inhibitory effect of melatonin was dose-dependent, with an IC₅₀ value of about 0.1 mM. However, a significant inhibition of enzyme activity (> 22%) was observed at 1 nM melatonin which is in the range of the physiological serum concentration of the hormone at night. The inhibitory effect of melatonin was observed exclusively in the presence of Ca⁺⁺. Results suggest a new and important role of the pineal hormone melatonin on central nervous system processes, i.e., by modulating NO production.     

Activation of MT(2) melatonin receptors in rat suprachiasmatic nucleus phase advances the circadian clock

The aim of this study was to identify the melatonin receptor type(s) (MT(1) or MT(2)) mediating circadian clock resetting by melatonin in the mammalian suprachiasmatic nucleus (SCN). Quantitative receptor autoradiography with 2-[(125)I]iodomelatonin and in situ hybridization histochemistry, with either (33)P- or digoxigenin-labeled antisense MT(1) and MT(2) melatonin receptor mRNA oligonucleotide probes, revealed specific expression of both melatonin receptor types in the SCN of inbred Long-Evans rats. The melatonin receptor type mediating phase advances of the circadian rhythm of neuronal firing rate in the SCN slice was assessed using competitive melatonin receptor antagonists, the MT(1)/MT(2) nonselective luzindole and the MT(2)-selective 4-phenyl-2-propionamidotetraline (4P-PDOT). Luzindole and 4P-PDOT (1 nM-1 microM) did not affect circadian phase on their own; however, they blocked both the phase advances (approximately 4 h) in the neuronal firing rate induced by melatonin (3 pM) at temporally distinct times of day [i.e., subjective dusk, circadian time (CT) 10; and dawn, CT 23], as well as the associated increases in protein kinase C activity. We conclude that melatonin mediates phase advances of the SCN circadian clock at both dusk and dawn via activation of MT(2) melatonin receptor signaling.     

Analysis of the pathways of nitric oxide utilization in mitochondria

The regulatory role that mitochondria play in cell dysfunction and cell-death pathways involves the concept of a complex and multisite regulation of cellular respiration and energy production signaled by cellular and intercellular messengers. Hence, the role of nitric oxide, as a physiological regulator acting directly on the mitochondrial respiratory chain acquires further relevance. This article provides a survey of the major regulatory roles of nitric oxide on mitochondrial functions as an expression of two major metabolic pathways for nitric oxide consumption: a reductive pathway, involving mitochondrial ubiquinol and yielding nitroxyl anion and an oxidative pathway involving superoxide anion and yielding peroxynitrite. The modulation of the decay pathways for nitrogen-and oxygen-centered radicals is further analyzed as a function of the redox transitions of mitochondrial ubiquinol. The interplay among these redox processes and its implications for mitochondrial function is discussed in terms of the mitochondrial steady-state levels (and gradients) of nitric oxide and superoxide anion.