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Journal of Pineal Research

Evidence that membrane-bound G protein-coupled melatonin receptors MT1 and MT2 are not involved in the neuroprotective effects of melatonin in focal cerebral ischemia

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Article Details

Authors

Ulkan Kilic, Bayram Yilmaz, Milas Ugur, Adnan Yüksel, Russel J. Reiter, Dirk M. Hermann, Ertugrul Kilic

Journal

Journal of Pineal Research

DOI

10.1111/j.1600-079x.2011.00932.x

Table of Contents

Abstract

Introduction

Materials And Methods

Animals

Animal Surgery

Western Blots

Statistics

Results

Discussion

Infarct Volume

Phosphorylated JNK1/2

Acknowledgements

Abstract

Introduction

Melatonin is synthesized and released into the circulation and especially into cerebrospinal fluid by the pineal gland in a circadian rhythm [1]. Its functions primarily depend in part on G(i) protein-coupled metabotropic receptors. To date, two types of melatonin membrane receptors have been characterized in mammals, the MT1 (Mel1a) and MT2 (Mel1b), which are expressed in many cells throughout the organism, including in the central nervous system [2, 3]. Melatonin plays an important role in the regulation of many physiologic functions, including as a circadian modulator, as an anti-inflammatory agent, and in the synchronization of seasonal reproductive rhythms [1, 4, 5]. Apart from its receptor-mediated metabolic actions, melatonin is a powerful antioxidant that scavenges the highly toxic hydroxyl radical as well as other radicals that initiate lipid peroxidation, protein oxidation, and DNA damage [6–9]. As melatonin synthesis and secretion decrease markedly during aging [1, 10, 11], a relative melatonin deficiency may be implicated in the pathophysiology of age-related oxidative neurodegenerative conditions, such as stroke. We and others have previously shown that the neuromolecule melatonin, because of its small molecular size and high lipophilicity, possesses excellent blood–brain barrier permeability, has minimal side effects in humans [12–14], and reduces brain injury in mouse [15–21] and rat [22–25] models of ischemic stroke. Furthermore, it has been found to be particularly suitable as an add-on treatment to thrombolytic drugs [16, 18]. Melatonin reduces the activity of inducible nitric oxide synthase (iNOS) [18] and nitric oxide (NO) production [24] after permanent or transient focal cerebral ischemia. When produced in high concentrations, NO causes lipid peroxidation especially after it couples with the superoxide anion to produce peroxynitrite, depletes cellular energy stores via disruption of mitochondrial enzymes, and damages nucleic acids [26]. Following transient focal cerebral ischemia, melatonin elevates Bcl-XL level and reduces caspase-3 activity and apoptotic cell death in ischemic brain tissue [16, 18]. Furthermore, melatonin inhibits a zinc-dependent circadian rhythm, and many of its peripheral actions are mediated via membrane MT1 and MT2 receptors. Apart from its metabolic functions, melatonin is a potent neuroprotective molecule owing to its antioxidative actions. The roles of MT1 and MT2 in the neuroprotective effects of melatonin and cell signaling after cerebral ischemia remain unknown. With the use of MT1 and MT2 knockout (mt1/2)/)) mice treated with melatonin, we evaluated brain injury, edema formation, inducible nitric oxide synthase (iNOS) activity, and signaling pathways, including CREB, ATF-1, p21, Jun kinase (JNK)1/2, p38 phosphorylation, resulting from ischemia/reperfusion injury. We show that the infarct volume and brain edema do not differ between mt1/2)/) and wild-type (WT) animals, but melatonin treatment decreases infarct volume in both groups and brain edema in WT animals after middle cerebral artery occlusion. Notably, melatonin s neuroprotective effect was even more pronounced in mt1/2)/) animals compared to that in WT animals. We also demonstrate that melatonin treatment decreased CREB, ATF-1, and p38 phosphorylation in both mt1/2)/) and WT mice, while p21 and JNK1/2 were reduced only in melatonin-treated WT animals in the ischemic hemisphere. Furthermore, melatonin treatment lowered iNOS activity only in WT animals. We provide evidence that the absence of MT1 and MT2 has no unfavorable effect on ischemic brain injury. In addition, the neuroprotective effects of melatonin appear to be mediated through a mechanism independent of its membrane receptors. The underlying mechanism(s) should be further studied using selective melatonin receptor agonists and antagonists. Ulkan Kilic1, Bayram Yilmaz2, Milas Ugur2, Adnan Yüksel1, Russel J. Reiter3, Dirk M. Hermann4 and Ertugrul Kilic2 1Department of Medical Biology, Faculty of Medicine, Bezmialem Vakif University, Istanbul, Turkey; 2Department of Physiology, Faculty of Medicine, Yeditepe University, Istanbul, Turkey; 3Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, TX, USA; 4Department of Neurology, University Hospital Essen, Essen, Germany Key words: cell signaling, cerebral ischemia, melatonin, melatonin receptors, stroke Address reprint requests to Ertugrul Kilic, Department of Physiology, Faculty of Medicine, Yeditepe University, TR-34755 Istanbul, Turkey. E-mails: kilic44@yahoo.com; ertugrul.kilic@ yeditepe.edu.tr Received June 27, 2011; Accepted August 8, 2011 M o le cu la r, B io lo g ic al ,P h ys io lo g ic al a n d C li n ic al A sp ec ts metalloprotease, endothelin converting enzyme-1 (ECE-1) [15], and matrix metalloproteinase-9 (MMP-9) [21] contributing to vascular dysfunction and damage after stroke [27, 28]. In addition, acute or prophylactic melatonin treatment increases Akt phosphorylation, and prophylactic treatment of melatonin activates (MAP) kinase/extracellular-regulated kinase (ERK)-1/-2 and Jun kinase (JNK)-1/-2 after transient focal cerebral ischemia [17]. Moreover, melatonin promotes neuronal survival, neurogenesis, and motor recovery as late as 30 days after ischemic stroke [19]. In this context, the mechanisms of the antioxidant actions of melatonin are complex and not solely restricted to free radical scavenging. Although administration of the melatonin receptor antagonist luzindole just prior to ischemia did not affect behavioral or histologic outcomes [29], in fact, the roles of melatonin receptors and the cytosolic signal transduction pathways, which may mediate melatonin s neuroprotection especially in terms of stimulating antioxidative enzymes [30, 31], remained largely unknown. To investigate these actions, herein, we subjected both WT and mt1/2)/) mice to 90 min of intraluminal middle cerebral artery (MCA) occlusion and examined the roles of melatonin receptors MT1 and MT2 and add-on treatment of melatonin after stroke. Twenty-four hours after ischemia, we analyzed infarct volume, brain edema as well as signal transduction factors, including the activation of iNOS, 3,5- cyclic adenosine monophosphate response element-binding protein (pCREB), p21, JNK-1/-2, and p38, which play significant roles in the pathogenesis of ischemic brain injury [17, 32–34].

Materials and methods

Animals

Experiments were performed in accordance with National Institutes of Health (NIH) guidelines for the care and use of laboratory animals and approved by local government authorities (Ethical Committee of the Yeditepe University). All animals were held under a constant 12:12-hr light/darkness regimen (lights on daily at 07.00 hr). In this study, male congenic MT1 and MT2 knockout mice (mt1/2)/)) produced on a C3H genetic background [35] together with wild-type C3H/HeN [denominated as wild type (WT)] control mice were used. The founder MT1 and MT2 knockout mice were kindly provided by Drs Reppert and Weaver of the University of Massachusetts Medical School, and wild-type C3H/HeN mice were purchased from Harlan Laboratories (Itingen, Switzerland). Adult male MT1 and MT2 knockout (mt1/2)/)) and WT mice weighing 22–26 g were randomly assigned to one of four groups, including vehicle-treated WT (i) or mt1/2)/), (ii) (n = 8 and 9, respectively) and melatonintreated WT, (iii) or mt1/2)/), and (iv) (n = 9 and 7, respectively) animals, which were submitted to focal cerebral ischemia and subsequently treated with intraperitoneal injection of vehicle (100 lL isotonic sodium chloride/5% ethanol) or melatonin (4 mg/kg, dissolved in 0.9% sodium chloride/5% ethanol) immediately after reperfusion.

Animal surgery

Animals were anesthetized with 1% isofluorane (30% O2 remainder N2O). Rectal temperature was maintained between 36.5 and 37.0 C using a feedback-controlled heating system (MAY Instruments, Ankara, Turkey). During the experiments, cerebral blood flow was measured by laser Doppler flow (LDF) recordings using a flexible 0.5-mm fiber optic probe (Perimed, Stockholm, Sweden), which was attached to the intact skull overlying the MCA territory (2 mm posterior/6 mm lateral from bregma). LDF changes were monitored up to 30 min after the onset of reperfusion. Focal cerebral ischemia was induced using an intraluminal filament technique [32]. A midline neck incision was made, and the left common and external carotid arteries were isolated and ligated. A microvascular clip (FE691; Aesculap, Tuttlingen, Germany) was temporarily placed on the internal carotid artery. A 8-0 nylon monofilament (Ethilon; Ethicon, Norderstedt, Germany) coated with silicon resin (Xantopren; Bayer Dental, Osaka, Japan; diameter of the coated thread: 180–190 lm) was introduced through a small incision into the common carotid artery and advanced 9 mm distal to the carotid bifurcation for MCA occlusion. Ninety minutes after induction of ischemia, reperfusion was initiated by withdrawal of monofilament. Thirty minutes after reperfusion onset, wounds were closed with suture, before anesthesia was discontinued. Twenty-four hours after MCA occlusion, animals were deeply anesthetized with 4% isofluorane (30% O2, remainder N2O) and decapitated. Brains were removed, frozen on dry ice, and cut on a cryostat into coronal 18-lm sections that were used for studying infarct volume and brain swelling. From the same animals, tissue samples were taken from the MCA territory (striatum and overlying parietal cortex) both ipsilateral and contralateral to the stroke for protein expression studies. Analysis of infarct volume and brain swelling Coronal brain sections from five equidistant brain levels, 2 mm apart, were stained with cresyl violet according to a standard protocol. On the sections, the border between infarcted and noninfarcted tissues was outlined using an image analysis system (Image J; National Institute of Health, Bethesda, MD, USA), and the area of infarction was assessed by subtracting the area of the nonlesioned ipsilateral hemisphere from that of the contralateral side. The volume of infarction was calculated by integration of these lesion areas. Edema was calculated as the volume difference between the ischemic and the nonischemic hemisphere and expressed as a percentage of the intact hemisphere.

Western blots

Brain tissue samples were harvested from ischemic and nonischemic striatum and overlying cortex of ischemic WT and MT1 and MT2 knockout mice. Tissue samples belonging to the same group were pooled, homogenized, and treated with protease inhibitor cocktail and phospha- tase inhibitor cocktail. The protein concentration was measured using Quibit Fluoremeter according to the manufacturer s protocol (Invitrogen, Life Technologies Corporation, Carlsbad, CA, USA). Equal amounts of protein (20 lg) were size-fractionated by 4–12% NuPAGE electrophoresis then transferred to polyvinylidene fluoride membrane (PVDF) using iBlot Dry Blotting System (Invitrogen, Life Technologies Corporation). Membranes were blocked in 5% nonfat milk in 50 mm Tris-buffered saline containing 0.1% Tween (blocking solution) for 1 hr at room temperature, washed in Trisbuffered saline containing 0.1% Tween (TBS-T), and incubated overnight with rabbit polyclonal anti-pp38 (sc-17852; Santa Cruz Biotechnology, Biocem, Istanbul, Turkey), goat polyclonal anti-pJNK (sc-12882; Santa Cruz Biotechnology), rabbit polyclonal anti-pp21 (sc-20220; Santa Cruz Biotechnology), rabbit polyclonal anti-NOS-2 (iNOS) (sc-650; Santa Cruz Biotechnology), and rabbit monoclonal anti-pCREB (9198; Cell Signaling, Sacem, Istanbul, Turkey) antibody. Rabbit monoclonal antipCREB antibody also detected the phosphorylated form of the CREB-related protein ATF-1 (p35). Each antibody was diluted 1:1000 directly in blocking solution. The following day, the membranes were first washed in TBS-T and incubated with peroxidase-conjugated goat anti-rabbit (Amersham, GE Health Care UK Limited, Buckinghamshire, England) and donkey anti-goat (sc-2033; Santa Cruz Biotechnology) antibody, diluted 1:5000 in blocking solution for 1 hr at room temperature. Blots were performed at least three times. Protein loading was controlled by stripping and reprobing the blots with rabbit polyclonal anti-b-actin antibody (4967; Cell Signaling). The blots were developed using ECL-Advanced Western Blotting Detection kit (Amersham, GE Health Care UK Limited) and visualized by the MF-ChemiBIS (DNR, Medsantek, Istanbul, Turkey). Protein levels were analyzed densitometrically using the ImageJ program and corrected with values determined on b-actin blots and expressed as relative values compared with WT mice.

Statistics

For statistical data comparisons, a standard software package (SPSS 18 for Windows; SPSS Inc., Chicago, IL, USA) was used. Differences between groups were analyzed by one-way ANOVA, followed by least significant differences tests. All values are given as mean ± S.E.M. with n values, indicating the number of different animals analyzed (n = 7–9 animals per group). P values <0.05 are considered significant. *P < 0.05/**P < 0.01 compared with vehicle-treated WT mice. #P < 0.05; ##P < 0.01 compared with vehicle-treated MT1/MT2)/) mice. §P < 0.05 compared with melatonin-treated WT mice. Values are given as mean ± S.E.M.

Results

To ensure the reproducibility of ischemia and to evaluate possible effects of melatonin and its receptors (MT1 and MT2) on brain hemodynamics during and after stroke, we analyzed LDF recordings above the core of the MCA territory. The MCA occlusion resulted in a decrease in LDF levels to approximately 15% of preischemic values (Fig. 1A). After reperfusion, blood flow rapidly resumed, reaching levels 15–60% above baseline. Although blood flow values during MCA occlusion and reperfusion were not statistically significant between animal groups, the mean LDF values during reperfusion were higher in melatonin-treated WT (approximately 42% above preischemic baseline) and mt1/2)/) (approximately 63% above baseline) animals than vehicle-treated WT (approximately 8% above baseline) and mt1/2)/) (approximately 37% above baseline) animals (Fig. 1A). Brain infarcts and ischemic brain edema (swelling) were morphologically evaluated by cresyl violet staining at 24 hr after 90 min MCA occlusion. In WT animals, reproducible brain infarcts were noted (Fig. 1B), which were associated with brain swelling (Fig. 1C). In the vehicle-treated animals, infarct volume did not differ betweenWT andmt1/2)/) mice, and they were 45.6 ± 7 and 39.7 ± 4 mm3, respectively. Melatonin-treated groups exhibited a significant (P < 0.01) infarct reduction by about 50% to 26.5 ± 3 and by about 75%to12.2 ± 3 mm3inmelatonin-treatedWTandmt1/2)/) animals, respectively (Fig. 1B). Although melatonin treatment reduced infarct volume significantly in both WT and mt1/2)/) groups, melatonin treatment reduced infarct volume more strongly in melatonin-treated mt1/2)/) than WT animals (P < 0.05) (Fig. 1B). Ischemic brain edema, expressed as percentage swelling of the ipsilateral hemisphere, was 7.21 ± 2% and 5.36 ± 1% of the contralateral hemisphere in vehicletreated WT and mt1/2)/) groups, respectively (Fig. 1C). Treatment with melatonin reduced brain edema to 3.16 ± 1% in WT and to 3.88 ± 1% in mt1/2)/) animals, but this effect was significant only in WT animals (P < 0.05). To characterize effects of melatonin and its receptor (MT1 and MT2) deactivation on intracellular signaling processes, tissue lysates from ischemic (ipsilateral) and nonischemic (contralateral) hemispheres were analyzed. Besides iNOS accumulation, primary antibodies detecting only phosphorylated, i.e., activated signaling factors were used and CREB, ATF-1, p21, JNK1/2, and p38 phosphorylation were evaluated and summarized in Fig. 2. In the nonischemic contralateral hemisphere, except pCREB (*P < 0.05) and pATF-1 (*P < 0.05) in vehicle-treated mt1/2)/) and iNOS (#P < 0.01 and §P < 0.05) in melatonin-treated mt1/2)/) animals, no statistically significant difference was observed between animal groups (Fig. 2). In the ischemic hemispheres, no significant difference was observed between WT and mt1/2)/) animals among these activated proteins (Fig. 2). Melatonin treatment significantly reduced accumulation of iNOS (*P < 0.01 and §P < 0.05), pCREB (*P < 0.05), pATF-1 (*P < 0.01), p-p21 (*P < 0.05), pJNK1/2 (*P < 0.01), and p-p38 (*P < 0.01) in WT animals (Fig. 2). Among these proteins, only pCREB (#P < 0.01), pATF-1 (#P < 0.05), and p-p38 (#P < 0.01) were significantly reduced in melatonin-treated mt1/2)/) animals. Our data indicate that melatonin may regulate iNOS, JNK1/2, and p-p21 through MT1 or MT2 receptors after focal cerebral ischemia (Fig. 2). *Compared with vehicle-treated WT mice. #Compared with vehicle-treated mt1/2)/) mice. §Compared with melatonin-treated WT mice.

Discussion

Based on previous findings showing that melatonin reduces ischemic injury following focal cerebral ischemia in rats [22–25] and mice [15–21], we made use of mt1/2)/) mice to examine the roles of G protein-coupled MT1 and MT2 receptors, of which some mediate melatonin s actions, in the development of brain injury and signaling pathways following 90 min of MCA occlusion. Brain perfusion was monitored by laser Doppler flowmetry. To assess the therapeutic potential of melatonin in the absence of its receptors MT1 and MT2, 4 mg/kg melatonin was administered immediately after reperfusion. Our data show for the first time that melatonin receptors MT1 and MT2 do not play a significant role in the development of brain injury after focal cerebral ischemia. Indeed, similar infarct volume and brain edema were observed in both mt1/2)/) and WT animals. In addition, melatonin treatment was neuroprotective not only in WT but also in mt1/2)/) mice. However, the reduction in infarct volume was significantly stronger in melatonin-treated mt1/2)/) mice as compared with melatonin-treated WT animals. Reduced brain edema was also observed in both groups of animals after melatonin treatment. However, it was only statistically significant in melatonin-treated WT animals. Based on our findings, the absence of melatonin receptors MT1 and MT2 does not lead to an unfavorable effect on the development of brain injury induced by MCA occlusion and to a loss of melatonin responsiveness. However, possible unfavorable side effects of melatonin receptors on neuronal injury should be further analyzed because significantly reduced infarct volume was observed in melatonin-treated mt1/2)/) mice. In the vascular system, opposite effects of melatonin receptors have been observed, in which the activation of MT1 and MT2 receptors stimulate vasoconstriction and vasodilation, respectively [36]. However, based on earlier observations, melatonin has beneficial effects on the vasculature under conditions in which cerebral hemodynamics are compromized, i.e., an increase in cerebral blood flow (CBF) [23] and an improvement of CBF autoregulation were observed [37]. In a previous study, we observed that melatonin inhibits ECE-1, a zincdependent metalloprotease produced by endothelial and smooth muscle cells; this agent cleaves inactive endothelin precursors to produce mature endothelin-1 [15]. Endothelin-1 is a highly potent endogenous vasoconstrictor, which contributes to vascular dysfunction after stroke

Infarct volume

Brain swelling 0 2 4 6 8 10 12 P er ce n t o f co n tr o l C re sy l v io le t st ai n in g * WT vehicle WT melatonin MT1/MT2–/– vehicle MT1/MT2–/– melatonin Laser Doppler flow 0 50 100 150 200 250 120 (min)900 P er ce n t o f co n tr o l (A) (B) (C) WT vehicle WT melatonin MT1/MT2–/– vehicle MT1/MT2–/– melatonin WT vehicle WT melatonin MT1/MT2–/– vehicle MT1/MT2–/– melatonin 0 10 20 30 40 50 60 ## § ** WT vehicle WT melatonin MT1/MT2–/– vehicle MT1/MT2–/– melatonin In fa rc t vo lu m e (m m 3 ) Fig. 1. (A) Laser Doppler flow (LDF) recordings during and after 90 min of intraluminal middle cerebral artery (MCA) occlusion in vehicle-treated wild type (WT), vehicle-treated MT1/MT2)/) (knockout), melatonin-treated WT, and melatonin-treated MT1/ MT2)/) animals. Note that LDF values after reperfusion are moderately higher in animals receiving melatonin than vehicle treatment. (B) and (C) brain edema assessed using cresyl violet–stained brain sections, analyzed 24 hr after 90 min of MCA occlusion. Note that infarct volume and brain edema do not differ between vehicle-treated animal groups after focal cerebral ischemia. Melatonin treatment decreases infarct volume in both groups. Notably, melatonin s neuroprotective effect was even more pronounced in MT1/MT2)/) mice compared to melatonin-treated WT animals. Effect of melatonin on brain edema was only significant in melatonin-treated WT animals. *P < 0.05 compared with vehicle-treated WT mice. *P < 0.05/**P < 0.01 compared with vehicle-treated WT mice. ##P < 0.01 compared with vehicletreated MT1/MT2)/) mice. §P < 0.05 compared with melatonintreated WT mice. Values are given as mean ± S.E.M. (n = 7–9 animals per group). Distance bar = 2 mm. [28]. In humans, endothelin-1 is implicated in the evolution of arterial hypertension, stimulates platelet aggregation, and also reinforces the formation of reactive oxygen species [38]. Furthermore, melatonin inhibits MMP-9 [21] contributing to vascular damage after stroke [28]. Although LDF does not provide accurate or precise measurements of absolute regional CBF values, it provides noninvasive, instantaneous, and continuous measurement of microcirculatory blood flow in a small tissue sample [39]. In this study, to evaluate possible effects of melatonin and its receptors (MT1 and MT2) on brain hemodynamics during and after stroke, we analyzed CBF by LDF using a flexible optic probe, which was attached to the intact skull overlying the MCA territory. Here, we observed moderate increase in CBF in both melatonin-treated mt1/2)/) and WT animals. In addition, CBF was slightly higher in vehicle-treated mt1/2)/) mice than vehicle-treated WT animals. In fact, this issue is a matter of further research, in which regional CBF would be measured precisely by autoradiographic techniques [40]. The highest level of melatonin is found in the mitochondria [41], and it has previously been shown that melatonin inhibits mitochondrial permeability transition pore formation [42, 43], diminishes cytochrome c release from the

Phosphorylated JNK1/2

injured mitochondria [44, 45], prevents caspase-3 activation [16, 18, 45], reduces DNA fragmentation [21], inhibits dissociation of pBad from 14-3-3. [45], attenuates MMP-9 [21], and increases Bcl-2 [45] and Bcl-XL [18] after focal cerebral ischemia. In addition, the neuroprotective action of melatonin is also mediated through the increased phosphorylation of the PI3-K/Akt survival pathway [17, 18], JNK-1/-2 [17], Raf-1, MEK1/2, and ERK1/2 and the downstream targets, including Bad and 90-kDa ribosomal S6 kinase [45]. Although G protein-coupled MT1 and MT2 melatonin receptors are expressed in neurons of the mammalian brain including in humans, relatively little is known about the influence of native MT1 and MT2 melatonin receptors on neuronal melatonin signaling in the ischemic conditions in vivo. Herein, we examined the activation of cytosolic cell signaling after transient focal cerebral ischemia in both mt1/ 2)/), produced on a C3H/HeN genetic background and WT animals by using Western blots for the phosphorylated, i.e., signaling factors CREB, ATF-1, p21, JNK-1/2, and p38. On the basis of in vitro findings, melatonin decreases CREB phosphorylation through activation of MT1 and MT2 receptors [47]. According to the effects of melatonin on the phosphorylation of CREB, we have also analyzed CREB phosphorylation. In contrast to hitherto studies, we observed that melatonin decreases phosphorylation of CREB and ATF-1 transcription factors not only in WT animals but also in mt1/2)/) animals, indicating MT1- and MT2 receptor-independent effects of melatonin on CREB and ATF-1 activations. Furthermore, decreased phosphorylation of p38 was also observed in both melatonin-treated WT and mt1/2)/) animals, which is strongly activated in injured neurons [32] and contributes neuronal injury. In addition, receptor-dependent phosphorylation of JNK-1/-2 was observed in melatonin-treated animals. Melatonin reduced the phosphorylation of JNK-1/-2 only in WT control animals, indicating receptor-dependent inhibition of these pathways. This is similar to some extent to recently reported action of melatonin on JNK-1/-2, in whichmelatonin decreased the phosphorylation of JNK-1/-2 after hepatic ischemia and reperfusion [48]. On the other hand, it has also been shown that melatonin stimulates JNK phosphorylation in cell culture [49]. We have also observed elevated phosphorylation of JNK-2 after focal cerebral ischemia [17]. However, in the aforementioned study, we have treated the animals with melatonin prophylactically, and proteins were isolated from whole ischemic brain [17]. Furthermore, we have observed that melatonin treatment decreases the phosphorylation of p21 in WT animals but not in mt1/2)/) animals. p53 contributes several cellular functions, such as the control of DNA damage, repair, and induction of apoptosis [50]. DNA damage leads to activation of the p53 protein, which further increases expression of proapoptotic Bax, GADD45, and major cell cycle regulator p21/WAF-1/CIP1. p21 is also an antiapoptotic protein and controls the integrity of genome and DNA repair. In fact, upregulation of p21 gene expression has been observed after permanent [51] and transient [50] focal brain ischemia. In addition, no correlation of p21 gene expression with cellular vulnerability observed, while it is upregulated especially in the perifocal areas [50]. p21 is considered also as a neuroprotective molecule. In this context, it has been suggested that p53 stimulates DNA repair or cell death through upregulation of Bax or p21, respectively [50, 52]. It has been shown earlier that melatonin reduces iNOS [18] (i.e., NOS 2) and NO [24] levels after permanent and transient focal cerebral ischemia. When produced in high concentrations, however, NO causes lipid peroxidation, depletes cellular energy stores via disruption of mitochondrial enzymes, and produces damage to nucleic acids [27]. iNOS is supposed to exacerbate ischemic damage after stroke, as it contributes to free radical stress via formation of nitrite and nitrate [53]. Inhibition of iNOS after focal cerebral ischemia has recently been shown by us also for another hypoxia-inducible factor, erythropoietin, which is also protective after brain ischemia [54]. Based on the present data, we have observed the significant inhibition of iNOS level in only melatonin-treated WT animals. In conclusion, the present report demonstrates that the melatonin receptors MT1 and MT2 do not aggravate or attenuate ischemic injury induced by MCA occlusion. Our data further indicate that intraperitoneal administration of melatonin reduces ischemic injury both in the presence and in the absence of the membrane-bound G protein-coupled receptors (MT1 and MT2). Furthermore, melatonin treatment improves infarct development more efficiently in MT1 and MT2 knockout animals. To elucidate the receptordependent neuroprotective efficacy of melatonin in detail, further studies should be performed using selective melatonin receptor agonist and antagonist in future. Finally, we present here signaling pathways playing roles in the pathogenesis of stroke; however, it should be mentioned that remarkable differences in cell signaling between C57BL and C3H mice were noted [55], of which researchers should be aware of.

Acknowledgements

We thank Drs Reppert and Weaver of the University of Massachusetts Medical School for providing us with the founder MT1 and MT2 knockout mice. This work was supported by European Molecular Biology Organization (EMBO) installation grant and The Turkish Academy of Sciences (TUBA/GEBIP).

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