Lipoxygenase-mediated pro-radical effect of melatonin via stimulation of arachidonicacid metabolismF. Radognaa, P. Sestilib, C. Martinellib, M. Paolillob, L. Paternosterc, M.C. Albertinic, A. Accorsic,G. Gualandid, L. Ghibellia,?aDipartimento di Biologia, Universita' di Roma Tor Vergata, via Ricerca Scientifica, 1 00133 Roma, ItalybIstituto di Ricerca sull'Attività Motoria, ItalycIstituto di Chimica Biologica, Universita' di Urbino Carlo Bo, ItalydDABAC, Universita' della Tuscia, Italya b s t r a c ta r t i c l ei n f oArticle history:Received 7 November 2008Revised 21 April 2009Accepted 12 May 2009Available online 19 May 2009Keywords:U937LipoxygenasePhospholipase A2Arachidonic acid5-HETECalmodulinWe have shown that melatonin immediately and transiently stimulates intracellular free radical productionon a set of leukocytes, possibly as a consequence of calmodulin binding. We show here that melatonin-induced ROS are produced by lipoxygenase (LOX), since they are prevented by a set of LOX inhibitors, and areaccompanied by increase of the 5-LOX product 5-HETE. LOX activation is accompanied by strong liberation ofAA; inhibition of Ca2+-independent, but not Ca2+-dependent, phospholipase A2 (PLA2), prevents bothmelatonin-induced arachidonic acid and ROS production, whereas LOX inhibition only prevents ROS,indicating that PLA2 is upstream with respect to LOX, as occurs in many signaling pathways. Chlorpromazine,an inhibitor of melatonin–calmodulin interaction, inhibits both ROS and arachidonic acid production, thuspossibly placing calmodulin at the origin of a melatonin-induced pro-radical pathway. Interestingly, it isknown that Ca2+-independent PLA2 binds to calmodulin: our results are compatible with PLA2 beingliberated by melatonin from a steady-state calmodulin sequestration, thus initiating an arachidonate signaltransduction. These results delineate a novel molecular pathway through which melatonin may participate tothe inflammatory response.© 2009 Elsevier Inc. All rights reserved.IntroductionMelatonin, a neuro-hormone principally produced by the pinealgland (Wurtman et al., 1964), is being receiving much attention as aregulator of organism homeostasis; its potential therapeutic use in thetreatment of many pathological conditions (Aggarwal and Shishodia,2006) is a maingoalof pharmacological research,sinceits natural originwouldensurelackofimportantsideeffects.Fromthispointofview,veryimportantwasthediscoveryofitsstronganti-oxidantability(Rodriguezetal.,2004),sincemanycriticalpathologiesareoriginated,orprogress,ininstances of insufficient radical scavenging. Melatonin biological activityresults also from its binding to high (plasma membrane MT1/MT2receptors) (Reppert et al.,1994) and low (quinone reductase, calmodu-lin) (Tan et al., 2007; Benítez-King et al., 1993) affinity targets withincells, in the nano- or micromolar range, respectively. Thus, on the onehand, MT1/MT2 engagement triggers a canonical intracellular signaltransduction (Radogna et al., 2007), whose results depend on thesignaling and enzymatic asset of each target cell; on the other hand,bindingtothecytosolicenzymatictargetsalterstheactivityoftheboundenzyme. It is not clear whether the high micromolar levels of melatoninrequired for quinone reductase (Tan et al., 2007) or calmodulin binding(Benítez-Kingetal.,1993)maybeactuallyreachedintheorganism,thusquestioning whether these interactions may have a physiologicalmeaning.Recently, increasing evidence showed that many non-neuroendo-crine tissues produce, and respond to, melatonin; this is especially truefor the white blood cells compartment (Carrillo-Vico et al., 2006), inwhich melatonin seems to play a role in controlling their number andfunctioning. This proposes melatonin as a possible modulator of theinflammatory/immune response. Indeed leucocytes possess all theenzymatic machinery necessary to synthesize melatonin from trypto-phan (Cubero et al., 2006) as well as the proper receptors (Carrillo-Vicoetal.,2004),thusbeinganautonomouscompartmentasfarasmelatoninresponses are concerned. Many evidence suggest that melatonin mayexert an anti-inflammatory effect; it has been proposed that melatoninanti-oxidant ability may contrast the onset and progression ofinflammation, phenomenon that implies production of reactive oxygenspecies and activation of pro-oxidant enzymes (Cuzzocrea and Reiter,2002). Since the anti-oxidant effect of melatonin improves at supra-physiological doses, this opens an important avenue for the pharmaco-logical use of melatonin as an anti-inflammatory adjuvant.Toxicology and Applied Pharmacology 238 (2009) 170–177? Corresponding author. Fax: +39 6 2023500.E-mail address: ghibelli@uniroma2.it (L. Ghibelli).0041-008X/$ – see front matter © 2009 Elsevier Inc. All rights reserved.doi:10.1016/j.taap.2009.05.011Contents lists available at ScienceDirectToxicology and Applied Pharmacologyjournal homepage: www.elsevier.com/locate/ytaap
