Melatonin and mitochondrial function during ischemia/reperfusion injury

Ischemia/reperfusion (IR) injury occurs in many organs and tissues, and contributes to morbidity and mortality worldwide. Melatonin, an endogenously produced indolamine, provides a strong defense against IR injury. Mitochondrion, an organelle for ATP production and a decider for cell fate, has been validated to be a crucial target for melatonin to exert its protection against IR injury. In this review, we first clarify the mechanisms underlying mitochondrial dysfunction during IR and melatonin’s protection of mitochondria under this condition. Thereafter, special focus is placed on the protective actions of melatonin against IR injury in brain, heart, liver, and others. Finally, we explore several potential future directions of research in this area. Collectively, the information compiled here will serve as a comprehensive reference for the actions of melatonin in IR injury identified to date and will hopefully aid in the design of future research and increase the potential of melatonin as a therapeutic agent.     

Effects of myocardial ischemia and reperfusion on mitochondrial function and susceptibility to oxidative stress

We investigated the effects of ischemia duration on the functional response of mitochondria to reperfusion and its relationship with changes in mitochondrial susceptibility to oxidative stress. Mitochondria were isolated from hearts perfused by the Langendorff technique immediately after different periods of global ischemia or reperfusion following such ischemia periods. Rates of O₂ consumption and H₂O₂ release with complex I- and complex II-linked substrates, lipid peroxidation, overall antioxidant capacity, capacity to remove H₂O₂, and susceptibility to oxidative stress were determined. The effects of ischemia on some parameters were time dependent so that the changes were greater after 45 than after 20 min of ischemia, or were significantly different to the nonischemic control only after 45 min of ischemia. Thus, succinate-supported state 3 respiration exhibited a significant decrease after 20 min of ischemia and a greater decrease after 45 min, while pyruvate malate-supported respiration showed a significant decrease only after 45 min of ischemia, indicating an ischemia-induced early inhibition of complex II and a late inhibition of complex I. Furthermore, both succinate and pyruvate malate-supported H₂O₂ release showed significant increases only after 45 min of ischemia. Similarly, whole antioxidant capacity significantly increased and susceptibility to oxidants significantly decreased after 45 min of ischemia. Such changes were likely due to the accumulation of reducing equivalents, which are able to remove peroxides and maintain thiols in a reduced state. This condition, which protects mitochondria against oxidants, increases mitochondrial production of oxyradicals and oxidative damage during reperfusion. This could explain the smaller functional recovery of the tissue and the further decline of the mitochondrial function after reperfusion following the longer period of oxygen deprivation. Received 18 May 2001; received after revision 17 July 2001; accepted 24 July 2001     

Lack of effect of antioxidant therapy during renal ischemia and reperfusion in dogs

Acute ischemic renal failure is of great clinical importance because of its frequent occurrence and the high mortality it causes. Recent observations indicate that reperfusion has its own dangers because of oxygenderived free radicals. To study this problem, ischemia was evoked in dogs in one kidney, by clamping the left renal artery for 45 min. This was followed by a 90-min period of reperfusion when diuresis, GFR, PAH clearance and sodium and potassium excretion were studied. Besides a control group (n=6), the following treatment groups were investigated. Allopurinol (n=7): 50 mg/kg for two days p.o. and 50 mg/kg in physiological saline infusion during the experiment; a small dose of SOD (n=6): 0.5 mg/kg in infusion, started 1 min before reperfusion and given continuously for 10 min; and a high dose of SOD (n=7): 5 mg/kg as above. In the first 15 min following reperfusion, the renal functions significantly worsened in all groups. Later on, the renal functions gradually improved and in the last period after reperfusion, GFR in the ischemic kidney was 64%, cPAH 59%, diuresis 60% and sodium and potassium excretion were 65% and 76%, respectively, of the basal values in the control group.Treatment with free radical scavengers did not cause any considerable changes in the renal functions. In some respects, the worst results were observed with low-level SOD treatment (cPAH, diuresis, as well as sodium and potassium excretion).At the end of reperfusion, there was a significant drop in sodium excretion by the right (intact circulation) kidney of the treated animals.     

Short cerebral ischemia and subsequent reperfusion and treatment with stobadine

Lipid peroxidation and activities of antioxidative enzymes were studied in the brain cortex after short (15 min) cerebral ischemia and reperfusion (10 min) in rats. Conjugated dienes (CD) and thiobarbituric acid-reactive substances (TBARS) were significantly elevated in the group of rats with ischemia followed by reperfusion in comparison to the ischemic animals. Superoxide dismutase (SOD) activity significantly increased in the group of animals with ischemia and reperfusion. No significant changes in the activities of glutathione peroxidase (GP) were observed. Stobadine administered before ischemia or before reperfusion decreased the level of TBARS. Stobadine probably prevents malondialdehyde (MDA) formation from hydroperoxide or might elevate the activity of aldehyde dehydrogenase. In contradiction to the findings after long-lasting (4 h) ischemia and subsequent reperfusion¹, no decrease in the concentration of CD or in the activity of SOD or GP was found.     

Changes in the cardiac glutathione status after ischemia and reperfusion

In the isolated and perfused rabbit heart ischemia induced a rapid decline of contractility, associated with a reduction of the content of tissue GSH with no significant changes in GSSG. Reperfusion induced a small recovery of contractility, a substantial release of total glutathione and a further decrease in the content of tissue GSH with a significant increase of tissue GSSG. Glutathione reductase and glutathione peroxidase activities were not affected by ischemia and reperfusion. This study suggests a possible role for glutathione in the determination of functional damage induced by myocardial ischemia and reperfusion.     

Changes in the cardiac glutathione status after ischemia and reperfusion

Summary In the isolated and perfused rabbit heart ischemia induced a rapid decline of contractility, associated with a reduction of the content of tissue GSH with no significant changes in GSSG. Reperfusion induced a small recovery of contractility, a substantial release of total glutathione and a further decrease in the content of tissue GSH with a significant increase of tissue GSSG. Glutathione reductase and glutathione peroxidase activities were not affected by ischemia and reperfusion. This study suggests a possible role for glutathione in the determination of functional damage induced by myocardial ischemia and reperfusion.This study was supported by a grant from Ministero Pubblica Istruzione and CNR Rome (no 8202331.56).     

Short cerebral ischemia and subsequent reperfusion and treatment with stobadine.

Lipid peroxidation and activities of antioxidative enzymes were studied in the brain cortex after short (15 min) cerebral ischemia and reperfusion (10 min) in rats. Conjugated dienes (CD) and thiobarbituric acid-reactive substances (TBARS) were significantly elevated in the group of rats with ischemia followed by reperfusion in comparison to the ischemic animals. Superoxide dismutase (SOD) activity significantly increased in the group of animals with ischemia and reperfusion. No significant changes in the activities of glutathione peroxidase (GP) were observed. Stobadine administered before ischemia or before reperfusion decreased the level of TBARS. Stobadine probably prevents malondialdehyde (MDA) formation from hydroperoxide or might elevate the activity of aldehyde dehydrogenase. In contradiction to the findings after long-lasting (4 h) ischemia and subsequent reperfusion, no decrease in the concentration of CD or in the activity of SOD or GP was found.     

Melatonin: presence and formation in invertebrates

In vertebrates, it is now clearly demonstrated that the pineal gland is implicated in conveying photoperiodic information via the daily pattern of melatonin secretion. Invertebrates, like vertebrates, use photoperiodic changes as a temporal cue to initiate physiological processes such as reproduction or diapause. How this information is integrated in invertebrates remains an unsolved question. Our review will be an attempt to evaluate the possible role of melatonin in conveying photoperiodic information in invertebrates. It is now well demonstrated in both vertebrates and invertebrates that melatonin as well as its precursors or synthesizing enzymes are present in various organs implicated in photoreceptive processes or in circadian pacemaking. Melatonin, serotonin or N-acetyltransferase have been found in the head, the eyes, the optic lobe and the brain of various invertebrate species. In some species it has also been shown that melatonin is produced rhythmically with high concentrations reached during the dark period. Moreover, the physiological effects of melatonin on various periodic processes such as rhythmic contractions in coelenterates, fissioning of asexual planarians or reproductive events in flies have been reported in the literature. All these results support the hypothesis (refs 36, 37) that melatonin is not solely a pineal hormone but that it may be an evolutionary conservative molecule principally involved in the transduction of photoperiodic information in all living organisms.     

Molecular mechanisms of glutamate-dependent neurodegeneration in ischemia and traumatic brain injury

Stroke and neurotrauma mediate neuronal death through a series of events that involve multiple interdependent molecular pathways. It has been suggested that these pathways are triggered following elevations in extracellular excitatory amino acids, primarily glutamate [1]. This report outlines mechanisms involving glutamate-mediated excitotoxicity with specific focus on (i) the role of Ca²⁺ in neurotoxicity, (ii) The concept of source specificity of neurotoxicity, (iii) the role of the ionotropic N-methyl-D-aspartate (NMDA)-subtype glutamate receptor and its associated submembrane molecules that may give rise to signaling specificity in excitotoxicity and (iv) the role of glutamate-mediated free-radical generation and associated cell death pathways. We also highlight a novel, peptide-based approach for uncoupling NMDA receptors from excitotoxicity in the rat central nervous system subjected to focal ischemia, thereby reducing stroke infarct volume and improving neurological functioning.Received 11 August 2003; received after revision 15 September 2003; accepted 17 September 2003     

Thyroid function parameters during a selenium repletion/depletion study in phenylketonuric subjects

Phenylketonuric (PKU) subjects have a limited supply of selenium (Se) in their phenylalanine-restricted diet. A Se repletion (1 g Se/kg/day)/depletion study was conducted in PKU children to determine the effect of Se on thyroid function parameters.The initial plasma Se concentration (mean±SD: 0.26±0.12 mol/L, p<0.00003, n=10) and glutathione peroxidase (GSH-Px) activity (140±58 U/L, p<0.00003, n=10) were significantly lower compared to agematched controls. After 14 weeks of supplementation, the plasma Se concentration (mean ±SD: 0.74±0.20 mol/L) normalized (normal range: 0.57–1.15 mol/L, mean ±SD: 0.76±0.13 mol/L, n=32) and remained stable thereafter during repletion. Plasma GSH-Px activity reached normal values after 18 weeks of supplementation (312±57 U/L; normal range: 238–492 U/L, mean ±SD: 345±54 U/L, n=32) and increased significantly for up to eight weeks thereafter (332±52 U/L). Individual and mean thyroid parameters were initially normal in all cases. The mean concentrations of plasma thyroxine (T₄: p<0.025), free T₄ (FT₄: p<0.01) and reverse triiodothyronine (rT₃: p<0.005) decreased to 75% of their initial value within three weeks of Se supplementation and remained stable thereafter, within a normal physiological range during selenium supplementation. They increased back to their initial values three weeks (T₄: p<0.05, FT₄: p<0.05) and six weeks (rT₃: p<0.025) respectively, after the end of the supplementation. In conclusion, Se supplementation modifies thyroid function parameters in Se-deficient PKU subjects most likely by an increase in activity of type I 5-deiodinase (5-DIase I).Preliminary results of this study were presented as posters on the 4th International Congress on Trace Elements in Medicine and Biology, Chamonix, France (1993) and the 4th Joint Meeting of the Lawson Wilkens Pediatric Endocrine Society and the European Society for Paediatric Endocrinology, San Francisco, California, Ped Res (1993) 33: S93 (abstract 537). Support in part by Research Grants of Nutricia BV, (The Netherlands).     

Chronic gestational exposure to ethanol impairs insulin-stimulated survival and mitochondrial function in cerebellar neurons

Chronic gestational exposure to ethanol has profound adverse effects on brain development. In this regard, studies using in vitro models of ethanol exposure demonstrated impaired insulin signaling mechanisms associated with increased apoptosis and reduced mitochondrial function in neuronal cells. To determine the relevance of these findings to fetal alcohol syndrome, we examined mechanisms of insulin-stimulated neuronal survival and mitochondrial function using a rat model of chronic gestational exposure to ethanol. In ethanol-exposed pups, the cerebellar hemispheres were hypoplastic and exhibited increased apoptosis. Isolated cerebellar neurons were cultured to selectively evaluate insulin responsiveness. Gestational exposure to ethanol inhibited insulin-stimulated neuronal viability, mitochondrial function, Calcein AM retention (membrane integrity), and GAPDH expression, and increased dihydrorosamine fluorescence (oxidative stress) and pro-apoptosis gene expression (p53, Fas-receptor, and Fas-ligand). In addition, neuronal cultures generated from ethanol-exposed pups had reduced levels of insulin-stimulated Akt, GSK-3β, and BAD phosphorylation, and increased levels of non-phosphorylated (activated) GSK-3β and BAD protein expression. The aggregate results suggest that insulin-stimulated central nervous system neuronal survival mechanisms are significantly impaired by chronic gestational exposure to ethanol, and that the abnormalities in insulin signaling mechanisms persist in the early postnatal period, which is critical for brain development. Received 21 January 2002; received after revision 28 February 2002; accepted 25 March 2002     

Bcl-2 family proteins and mitochondrial fission/fusion dynamics

Mitochondria are dynamic organelles and can undergo regulated fission/fragmentation to produce smaller organelles or, alternatively, can undergo fusion to produce tubular or net-like mitochondrial structures. Although some of the molecules that control mitochondrial fission and fusion are known, new molecules and pathways that control this process continue to be discovered, suggesting that this process is more complex than previously appreciated. In addition to their crucial role in the regulation of apoptosis, recent studies have implicated members of the Bcl-2 family in maintenance of the mitochondrial network. Here, we discuss the mechanisms governing mitochondrial fission/fusion and summarize current knowledge concerning the role of Bcl-2 family members in regulating mitochondrial fission/fusion dynamics.     

The relationship between membrane potential and ATP content in rat liver during ischemia

Summary Both ischemia and ethionine decreased the membrane potential and ATP content in rat liver. In ethioninetreated rat liver, ischemia brought about a further decrease in membrane potential without significant decrease in ATP content.     

Melatonin and cell death: differential actions on apoptosis in normal and cancer cells

Melatonin is a natural compound synthesized by a variety of organs. It has been shown to function as a cell-protective agent. Since 1994, when the first paper was published documenting the role of melatonin in apoptosis, the number of reports in this area has increased rapidly. Much of the research conducted falls into three major categories: first, the role of melatonin in inhibiting apoptosis in immune cells; second, the role of melatonin in preventing neuronal apoptosis and finally, the role of melatonin in increasing apoptotic cell death in cancer cells. The mechanisms whereby melatonin influences apoptosis have not clarified, although a number of mechanistic options have been suggested. Apoptotic cell death is a physiological phenomenon related to homeostasis and proper functioning of tissues and organs; however, a failure in the apoptotic program is related to a number of diseases. The participation of melatonin in apoptosis in numerous cell types and its potential importance in a variety of diseases such as immunodeficiency, neurodegeneration and cancer is summarized in this review.Received 14 November 2002; received after revision 16 January 2003; accepted 10 February 2003     

Scaffolding microdomains and beyond: the function of reggie/flotillin proteins

Reggie/flotillin proteins are considered to be components of lipid rafts and are commonly used as marker proteins for lipid microdomains. Yet almost a decade after their discovery, the function of reggies/ flotillins is still enigmatic. In this review we summarize the present state of knowledge on reggie/flotillin structure, localization and function, and discuss the role of the proteins in development and disease. Based on insights into reggie/flotillin function and by comparison with related proteins of the so-called SPFH (Stomatin/Prohibitin/Flotillin/HflK/C) protein family, including stomatin, podocin and prohibitin, we propose the existence of specific types of protein-defined microdomains which are sculpt by the clustering of individual SPFH proteins. As 'specialized rafts' similar to caveolae, these membrane domains provide platforms for the recruitment of multiprotein complexes. Since, under certain circumstances, reggie-2/flotillin-1 translocates to the nucleus, reggie/ flotillin microdomains are not only stable scaffolds but also dynamic units with their own regulatory functions.