Ana López

Extrapineal melatonin: analysis of its subcellular distribution and daily fluctuations

Abstract:  We studied the subcellular levels of melatonin in cerebral cortex and liver of rats under several conditions. The results show that melatonin levels in the cell membrane, cytosol, nucleus, and mitochondrion vary over a 24‐hr cycle, although these variations do not exhibit circadian rhythms. The cell membrane has the highest concentration of melatonin followed by mitochondria, nucleus, and cytosol. Pinealectomy significantly increased the content of melatonin in all subcellular compartments, whereas luzindole treatment had little effect on melatonin levels. Administration of 10 mg/kg bw melatonin to sham‐pinealectomized, pinealectomized, or continuous light‐exposed rats increased the content of melatonin in all subcellular compartments. Melatonin in doses ranging from 40 to 200 mg/kg bw increased in a dose‐dependent manner the accumulation of melatonin on cell membrane and cytosol, although the accumulations were 10 times greater in the former than in the latter. Melatonin levels in the nucleus and mitochondria reached saturation with a dose of 40 mg/kg bw; higher doses of injected melatonin did not further cause additional accumulation of melatonin in these organelles. The results suggest some control of extrapineal accumulation or extrapineal production of melatonin and support the existence of regulatory mechanisms in cellular organelles, which prevent the intracellular equilibration of the indolamine. Seemingly, different concentrations of melatonin can be maintained in different subcellular compartments. The data also seem to support a requirement of high doses of melatonin to obtain therapeutic effects. Together, these results add information that assists in explaining the physiology and pharmacology of melatonin.

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

Abstract:  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.

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.

Chronic melatonin treatment reduces the age‐dependent inflammatory process in senescence‐accelerated mice

Abstract:  It is hypothesized that, besides increased free radical production, aging is a process also related to inflammation. Thus, female and male senescence‐accelerated (SAMP8) and senescence‐resistant (SAMR1) mice of 5 and 10 months of age were studied to assess this hypothesis. Plasma from these mice was processed to determine nitric oxide (NO), and pro‐inflammatory [interleukin (IL)‐1β, IL‐2, interferon (IFN)‐γ, tumor necrosis factor (TNF)‐α, and granulocyte–macrophage colony‐stimulating factor] and anti‐inflammatory (IL‐4, IL‐5 and IL‐10) cytokines. The results show the presence of an age‐dependent increase in IFN‐γ and TNF‐α and a reduction in IL‐2 levels, with minor changes in the remaining cytokines. Moreover, age was associated with a significant increase in NO levels. Chronic melatonin administration between 1 and 10 months of age counteracted the age‐dependent production of pro‐inflammatory cytokines and NO, reducing them to the levels found at 5 months of age. Melatonin also reduced the levels of the anti‐inflammatory cytokines. The results of this study suggest the existence of an inflammatory process during aging and further support that melatonin behaves as an essential molecule against aging, for its anti‐inflammatory properties together with its antioxidative role reported elsewhere.

Melatonin and its brain metabolite N1‐acetyl‐5‐methoxykynuramine prevent mitochondrial nitric oxide synthase induction in parkinsonian mice

Melatonin prevents mitochondrial failure in models of sepsis through its ability to inhibit the expression and activity of both cytosolic (iNOS) and mitochondrial (i‐mtNOS) inducible nitric oxide synthases. Because Parkinsons disease (PD), like sepsis, is associated with iNOS induction, we assessed the existence of changes in iNOS/i‐mtNOS and their relation with mitochondrial dysfunction in the MPTP model of PD, which also displays increased iNOS expression. We also evaluated the role of melatonin (aMT) and its brain metabolite, N1‐acetyl‐5‐methoxykynuramine (AMK), in preventing i‐mtNOS induction and mitochondrial failure in this model of PD. Mitochondria from substantia nigra (SN) and, to a lesser extent, from striatum (ST) showed a significant increase in i‐mtNOS activity, nitrite levels, oxidative stress, and complex I inhibition after MPTP treatment. MPTP‐induced i‐mtNOS was probably related to mitochondrial failure, because its prevention by aMT and AMK reduced oxidative/nitrosative stress and restored complex I activity. These findings represent the first experimental evidence of a potential role for i‐mtNOS in the mitochondrial failure of PD and support a novel mechanism in the neuroprotective effects of aMT and AMK.

Cellular mechanisms involved in the melatonin inhibition of HT-29 human colon cancer cell proliferation in culture

Abstract:  The antiproliferative and proapoptotic properties of melatonin in human colon cancer cells in culture were recently reported. To address the mechanisms involved in these actions, HT‐29 human colon cancer cells were cultured in RPMI 1640 medium supplemented with fetal bovine serum at 37°C. Cell proliferation was assessed by the incorporation of [3H]‐thymidine into DNA. Cyclic nucleotide levels, nitrite concentration, glutathione peroxidase and reductase activities, and glutathione levels were assessed after the incubation of these cells with the following drugs: melatonin membrane receptor agonists 2‐iodo‐melatonin, 2‐iodo‐N‐butanoyl‐5‐methoxytryptamine, 5‐methoxycarbonylamino‐N‐acetyltryptamine (GR‐135,531), and the antagonists luzindole, 4‐phenyl‐2‐propionamidotetralin, and prazosin; the melatonin nuclear receptor agonist CGP 52608, and four synthetic kynurenines analogs to melatonin 2‐acetamide‐4‐(3‐methoxyphenyl)‐4‐oxobutyric acid, 2‐acetamide‐4‐(2‐amino‐5‐methoxyphenyl)‐4‐oxobutyric acid, 2‐butyramide‐4‐(3‐methoxyphenyl)‐4‐oxobutyric acid and 2‐butyramide‐4‐(2‐amino‐5‐methoxyphenyl)‐4‐oxobutyric acid. The results show that the membrane receptors are not necessary for the antiproliferative effect of melatonin and the participation of the nuclear receptor in this effect is suggested. Moreover, the antioxidative and anti‐inflammatory actions of melatonin, counteracting the oxidative status and reducing the production of nitric oxide by cultured HT‐29 cells seem to be directly involved in the oncostatic properties of melatonin. Some of the synthetic kynurenines exert higher antiproliferative effects than melatonin. The results reinforce the clinical interest of melatonin due to the different mechanisms involved in its oncostatic role, and suggest a new synthetic pathway to obtain melatonin agonists with clinical applications to oncology.

Improved mitochondrial function and increased life span after chronic melatonin treatment in senescent prone mice

We investigated whether chronic melatonin administration influences mitochondrial oxidative stress and life span in mice. Diaphragmatic mitochondria from female senescent prone (SAMP8) and senescent resistant (SAMR1) mice at 5 and 10 months of age were studied. Mitochondrial oxidative stress was determined by measuring the levels of lipid peroxidation, glutathione and glutathione disulfide, and glutathione peroxidase and reductase activities. Mitochondrial function was assessed by measuring the activity of the respiratory chain complexes and the ATP content. The results suggest that the age-dependent mitochondrial oxidative damage in the diaphragm of SAMP8 mice was accompanied by a reduction in the electron transport chain complex activities and in ATP levels. Furthermore, melatonin administration between 1 and 10 months of age normalized the redox and the bioenergetic status of the mitochondria and increased the ATP levels. Melatonin also increased both half-life and longevity, mainly in SAMP8 group. These results suggest an age-related increase in mitochondria vulnerability to oxidation in SAM mice at 10 months of age that was counteracted by melatonin therapy. The effects of melatonin on mitochondrial physiology probably underline the ability of the indoleamine to increase maximal life span in these animals.

Melatonin blunts the mitochondrial/NLRP3 connection and protects against radiation-induced oral mucositis

Mucositis is a common and distressing side effect of chemotherapy or radiotherapy that has potentially severe consequences, and no treatment is available. The purpose of this study was to analyze the molecular pathways involved in the development of oral mucositis and to evaluate whether melatonin can prevent this pathology. The tongue of male Wistar rats was subjected to irradiation (X‐ray YXLON Y.Tu 320‐D03 irradiator; the animals received a dose of 7.5 Gy/day for 5 days). Rats were treated with 45 mg/day melatonin or vehicle for 21 days postirradiation, either by local application into their mouths (melatonin gel) or by subcutaneous injection. A connection between reactive oxygen species, generating mitochondria and the NLRP3 (NLR‐related protein 3 nucleotide‐binding domain leucine‐rich repeat containing receptor‐related protein 3) inflammasome, has been reported in mucositis. Here, we show that mitochondrial oxidative stress, bioenergetic impairment and subsequent NLRP3 inflammasome activation are involved in the development of oral mucositis after irradiation and that melatonin synthesized in the rat tongue is depleted after irradiation. The application of melatonin gel restores physiological melatonin levels in the tongue and prevents mucosal disruption and ulcer formation. Melatonin gel protects the mitochondria from radiation damage and blunts the NF‐κB/NLRP3 inflammasome signaling activation in the tongue. Our results suggest new molecular pathways involved in radiotherapy‐induced mucositis that are inhibited by topical melatonin application, suggesting a potential preventive therapy for mucositis in patients with cancer.

The beneficial effects of melatonin against heart mitochondrial impairment during sepsis: inhibition of iNOS and preservation of nNOS.

While it is accepted that the high production of nitric oxide (NO˙) by the inducible nitric oxide synthase (iNOS) impairs cardiac mitochondrial function during sepsis, the role of neuronal nitric oxide synthase (nNOS) may be protective. During sepsis, there is a significantly increase in the expression and activity of mitochondrial iNOS (i‐mtNOS), which parallels the changes in cytosolic iNOS. The existence of a constitutive NOS form (c‐mtNOS) in heart mitochondria has been also described, but its role in the heart failure during sepsis remains unclear. Herein, we analyzed the changes in mitochondrial oxidative stress and bioenergetics in wild‐type and nNOS‐deficient mice during sepsis, and the role of melatonin, a known antioxidant, in these changes. Sepsis was induced by cecal ligation and puncture, and heart mitochondria were analyzed for NOS expression and activity, nitrites, lipid peroxidation, glutathione and glutathione redox enzymes, oxidized proteins, and respiratory chain activity in vehicle‐ and melatonin‐treated mice. Our data show that sepsis produced a similar induction of iNOS/i‐mtNOS and comparable inhibition of the respiratory chain activity in wild‐type and in nNOS‐deficient mice. Sepsis also increased mitochondrial oxidative/nitrosative stress to a similar extent in both mice strains. Melatonin administration inhibited iNOS/i‐mtNOS induction, restored mitochondrial homeostasis in septic mice, and preserved the activity of nNOS/c‐mtNOS. The effects of melatonin were unrelated to the presence or the absence of nNOS. Our observations show a lack of effect of nNOS on heart bioenergetic impairment during sepsis and further support the beneficial actions of melatonin in sepsis.

Dysfunctional Coq9 protein causes predominant encephalomyopathy associated with CoQ deficiency

Coenzyme Q10 (CoQ(10)) or ubiquinone is a well-known component of the mitochondrial respiratory chain. In humans, CoQ(10) deficiency causes a mitochondrial syndrome with an unexplained variability in the clinical presentations. To try to understand this heterogeneity in the clinical phenotypes, we have generated a Coq9 Knockin (R239X) mouse model. The lack of a functional Coq9 protein in homozygous Coq9 mutant (Coq9(X/X)) mice causes a severe reduction in the Coq7 protein and, as consequence, a widespread CoQ deficiency and accumulation of demethoxyubiquinone. The deficit in CoQ induces a brain-specific impairment of mitochondrial bioenergetics performance, a reduction in respiratory control ratio, ATP levels and ATP/ADP ratio and specific loss of respiratory complex I. These effects lead to neuronal death and demyelinization with severe vacuolization and astrogliosis in the brain of Coq9(X/X) mice that consequently die between 3 and 6 months of age. These results suggest that the instability of mitochondrial complex I in the brain, as a primary event, triggers the development of mitochondrial encephalomyopathy associated with CoQ deficiency.