Lipoxygenase-mediated pro-radical effect of melatonin via stimulation of arachidonic acid metabolism (original) (raw)
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Toxicology and Applied Pharmacology, 2009
Melatonin is a modified tryptophan with potent biological activity, exerted by stimulation of specific plasma membrane (MT1/MT2) receptors, by lower affinity intracellular enzymatic targets (quinone reductase, calmodulin), or through its strong anti-oxidant ability. Scattered studies also report a perplexing pro-oxidant activity, showing that melatonin is able to stimulate production of intracellular reactive oxygen species (ROS). Here we show that on U937 human monocytes melatonin promotes intracellular ROS in a fast (b 1 min) and transient (up to 5-6 h) way. Melatonin equally elicits its pro-radical effect on a set of normal or tumor leukocytes; intriguingly, ROS production does not lead to oxidative stress, as shown by absence of protein carbonylation, maintenance of free thiols, preservation of viability and regular proliferation rate. ROS production is independent from MT1/MT2 receptor interaction, since a) requires micromolar (as opposed to nanomolar) doses of melatonin; b) is not contrasted by the specific MT1/MT2 antagonist luzindole; c) is not mimicked by a set of MT1/MT2 high affinity melatonin analogues. Instead, chlorpromazine, the calmodulin inhibitor shown to prevent melatonin-calmodulin interaction, also prevents melatonin pro-radical effect, suggesting that the low affinity binding to calmodulin (in the micromolar range) may promote ROS production.
Mitochondrion, 2015
Extra-neurological functions of melatonin include control of the immune system and modulation of apoptosis. We previously showed that melatonin inhibits the intrinsic apoptotic pathway in leukocytes via stimulation of high affinity MT1/MT2 receptors, thereby promoting re-localization of the anti-apoptotic Bcl-2 protein to mitochondria. Here we show that Bcl-2 sequesters pro-apoptotic Bax into mitochondria in an inactive form after melatonin treatment, thus reducing cell propensity to apoptosis. Bax translocation and the anti-apoptotic effect of melatonin are strictly dependent on the presence of Bcl-2, and on the 5-lipoxygenase (5-LOX) metabolite 5-hydroxyeicosatetraenoic acid (5-HETE), which we have previously shown to be produced as a consequence of melatonin binding to its low affinity target calmodulin. Therefore, the anti-apoptotic effect of melatonin requires the simultaneous, independent interaction with high (MT1/MT2) and low (calmodulin) affinity targets, eliciting two independent signal transduction pathways converging into Bax sequestration and inactivation. MT1/MT2 vs. lipoxygenase pathways are activated by 10 −9 vs. 10 −5 M melatonin, respectively; the antiapoptotic effect of melatonin is achieved at 10 −5 M, but drops to 10 −9 M upon addition of exogenous 5-HETE, revealing that lipoxygenase activation is the rate-limiting pathway. Therefore, in areas of inflammation with increased 5-HETE levels, physiological nanomolar concentrations of melatonin may suffice to maintain leukocyte viability.
Melatonin: A pleiotropic molecule regulating inflammation
Biochemical Pharmacology, 2010
Melatonin is a neurohormone produced by the pineal gland that regulates sleep and circadian functions. Melatonin also regulates inflammatory and immune processes acting as both an activator and inhibitor of these responses. Melatonin demonstrates endocrine, but also paracrine and autocrine effects in the leukocyte compartment: on one side, leukocytes respond to melatonin in a circadian fashion; on the other side, leukocytes are able to synthesize melatonin by themselves. With its endocrine and paracrine effects, melatonin differentially modulates pro-inflammatory enzymes, controls production of inflammatory mediators such as cytokines and leukotrienes and regulates the lifespan of leukocytes by interfering with apoptotic processes. Moreover, its potent anti-oxidant ability allows scavenging of oxidative stress in the inflamed tissues. The interesting timing of pro-and anti-inflammatory effects, such as those affecting lipoxygenase activity, suggests that melatonin might promote early phases of inflammation on one hand and contribute to its attenuation on the other hand, in order to avoid complications of chronic inflammation. This review aims at giving a comprehensive overview of the various inflammatory pathways regulated by this pleiotropic hormone.
2005
Inflammation is a complex phenomenon involving multiple cellular and molecular interactions which must be tightly regulated. Cyclooxygenase-2 (COX) is the key enzyme that catalyzes the two sequential steps in the biosynthesis of PGs from arachidonic acid. The inducible isoform of COX, namely COX-2, plays a critical role in the inflammatory response and its over-expression has been associated with several pathologies including neurodegenerative diseases and cancer. Melatonin is the main product of the pineal gland with well documented antioxidant and immuno-modulatory effects. Since the action of the indole on COX-2 has not been previously described, the goal of the present report was to test the effect of melatonin on the activities of COX-2 and inducible nitric oxide synthase (iNOS), using lipopolysaccharide (LPS)-activated RAW 264.7 macrophages as a model. Melatonin and its metabolites, N1-acetyl-N2-formyl-5methoxykynuramine (AFMK) and N1-acetyl-5-methoxykynuramine (AMK), prevented COX-2 activation induced by LPS, without affecting COX-1 protein levels. The structurally related compound 6-methoxy-melatonin only partially prevented the increase in COX-2 protein levels induced by the toxin. Likewise melatonin prevented iNOS activation and reduced the concentration of products from both enzymes, PGE 2 and nitric oxide. Another endogenous antioxidant like N-acetyl-cysteine (NAC) did not reduced COX-2 significantly. The current finding corroborates a role of melatonin as an anti-inflammatory agent and, for the first time, COX-2 and iNOS as molecular targets for either melatonin or its metabolites AFMK and AMK. These anti-inflammatory actions seem not to be exclusively mediated by the free radical scavenging properties of melatonin. As a consequence, the present work suggests these substances as a new class of potential antiinflammatory agents without the classical side effects due to COX-1 inhibition. D
Melatonin reduces both basal and bacterial lipopolysaccharide-induced lipid peroxidation in vitro
Free Radical Biology and Medicine, 1995
The protective effect of melatonin against lipopolysaccharide (LPS)-induced oxidative damage was examined in vitro. Lung, liver, and brain malonaldehyde (MDA) plus 4-hydroxyalkenals (CHDA) concentrations were measured as indices of induced membrane peroxidative damage. Homogenates of brain, lung, and liver were incubated with LPS at concentrations of either 1, 10, 50, 200, or 400 &ml for 1 h and, in another study, LPS at a concentration of 400 &ml for either 0, 15, 30, or 60 min. Melatonin at increasing concentrations from 0.01-3 mM either alone or together with LPS (400 &ml) was used. Liver, brain, and lung MDA + 4-HDA levels increased after LPS at concentrations of 10, 50, 200 or 400 &ml; this effect was concentration-dependent.
Journal of Pineal Research, 2003
Abstract: We have investigated the action of melatonin against lipid peroxidation in membranes including brain homogenates (BH), brain and liver microsomes (MIC), and phosphatidylcholine (PC) liposomes, as well as its effect on the activity of pro-oxidant enzymes such as constitutive neuronal nitric oxide synthase (cnNOS), xanthine oxidase (XO) and myeloperoxidase (MPO). The liposomes were reconstituted by a dialysis method, lipid peroxidation was monitored using the thiobarbituric reactive substances (TBARS) method and enzyme activities were measured spectrophotometrically. The ascorbyl and hydroxyl free radicals were generated by the reaction of ascorbic acid + FeSO4 and H2O2 + FeCl2, respectively, and peroxynitrite using a mixture of NaNO2 in an alkaline medium. Melatonin protected against lipid peroxidation induced by distinct reactive oxygen species (ROS) in all membranes tested although with different potency, in the following order BH < MIC < PC. The K0.5 for enzyme inhibition by melatonin was determined for nNOS (2.0 ± 0.1 mm), for XO (0.8 ± 0.1 mm) and for MPO (0.063 ± 0.003 mm), the latter one with high affinity. Melatonin showed a weak effect as a nitrogen monoxide (NO) scavenger in the presence of sodium nitroprusside (NO donor) and low reactivity with 1,1–diphenyl-2-picryl hydrazyl (DPPH). These results demonstrate the antioxidant action of melatonin, principally that related to the activity of pro-oxidant enzymes such as XO and MPO.