Tropisetron upregulates cannabinoid CB1 receptors in cerebellar granule cells: Possible involvement of calcineurin

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    Tropisetron upregulagranule cells: Possibl

    a ezaizadMo

    f Medif Medi

    c Schoolal Med

    entarif r, Tehr


    granisetron at 1 nM10 M concentrations. Moreover, cannabinoid CB1 receptor expression

    B R A I N R E S E A R C H 1 4 1 7 ( 2 0 1 1 ) 1 8

    Ava i l ab l e on l i ne a t i enced i r ec t . comat mRNA and protein levels were investigated by real time PCR and western blotting,respectively and its functionality studied by measuring the secondary messenger cAMP inCGNs receiving tropisetron or granisetron.Results indicate that tropisetron, but not granisetron, significantly inhibits the phosphataseactivity of calcineurin, over-expresses the CB1 receptors at both transcriptional and proteinlevels, and reduces cAMP content.A R T I C L E I N F O A B S T R A C T

    Article history:Accepted 19 August 2011Available online 27 August 2011

    Tropisetron, a selective 5-HT3 receptor antagonist, is widely used to counteract chemotherapy-induced emesis. Some investigations describe disparate effects including immunomodulatoryproperties for tropisetron which may be mediated through immunophilincalcineurinpathway. Calcineurin, a phosphatase involved in immune system signaling, modulatesexpression of several genes, such as Cannabinoid type one (CB1) receptors. On the quest forits underlying mechanisms of action, this study aimed to investigate the effect of tropisetronon calcineurin activity and CB1 receptor expression and function in cerebellar granuleneurons (CGNs).The rat pup CGNswere used as highly calcineurin-rich and devoid of 5-HT3 receptor neuronalcells. Calcineurin activity was assessed in CGNs treated with tropisetron or the congener

    Keywords:TropisetronCannabinoid receptorCerebellar granule neuronsCalcineurinCellular and Molecular Research CentegDepartment of Medical Biotechnology, Corresponding author at: P.O. Box: 13145-78E-mail address: (S.EAbbreviations: CB1, cannabinoid type one;

    0006-8993/$ see front matter 2011 Elseviedoi:10.1016/j.brainres.2011.08.050an University of Medical Sciences, Tehran, Iranof Medical Sciences, Tarbiat Modares University, Tehran, IranDepartment of Medical Biotechnology,dDepartment of Immunology & InternWinnipeg, CanadaeDipartimento di Scienze Chimiche Alimtes cannabinoid CB1 receptors in cerebellare involvement of calcineurin

    Dehpoura, Gohar Fakhfourib,ehc, Jean-Eric Ghiad, Mohammad Seyedabadia,usavizadehf, Mehdi Forouzandehg, Shahram Ejtemaei Mehra,

    cine, Tehran University of Medical Sciences, Tehran, Irancine, Shahid Beheshti University of Medical Sciences, Tehran, Iranof Advanced Medical Technologies, Tehran University of Medical Sciences, Tehran, Iranicine Section of Gastroenterology, Faculty of Medicine, University of Manitoba, Manitoba,

    Farmaceutiche e Farmacologiche, Universit del Piemonte Orientale A. Avogadro, Novara, Italy

    www.e l sev i e r . com/ loca te /b ra i n resReza Rahimian , Ahmad RMohammad Reza KhorramAntonio Caldarellie, KazemaDepartment of Pharmacology, School obDepartment of Pharmacology, School oOur investigation shows that tropisetron targets calcineurin in a receptor-independent fashion.Tropisetron-induced CB1 receptor up-regulation might underlie many pharmacological aspectsof tropisetron unrelated to anti-emesis.

    2011 Elsevier B.V. All rights reserved.

    4 Tehran, Iran. Fax: +98 21 6640 2569.. Mehr).CGNs, cerebellar granule neurons

    r B.V. All rights reserved.

  • properties and taking into account the ability of calcineurin tocontrol CB1 expression (Kramer et al., 2003),we aimed to investi-gate possible effects of tropisetron on calcineurin activity andCB1 receptor expression as well as its secondary messenger,cAMP, content in primary cerebral granule neuron cultures.Their high content of calcineurin (Klee et al., 1998) makes cere-bellar granule cells ideal for investigating effects of tropisetronon calcineurin activity. The findings of this study would add toour understanding of novelmechanisms underlying pharmaco-logical actions of tropisetron.

    2. Results

    2.1. Effect of tropisetron on cell viability

    To determine optimal dose ranges of tropisetron and granise-tron for use throughout the experiment, effects of differentdoses of tropisetron (1 nM1 mM) or granisetron (1 nM1 mM)on cell viability were assessed usingMTTmethod. Both tropise-

    H 1 4 1 7 ( 2 0 1 1 ) 1 8setron and CB1 receptor agonists share various pharmacologicaleffects including anti-emetic (Scuderi, 2001) analgesic (Riering

    range of 1 nM10 M did not affect cell viability after 4 DIV,while at 100 M1 mM significantly reduced viable cells1. Introduction

    Tropisetron is a selective 5-HT3 receptor antagonist widely usedto counteract chemotherapy-induced emesis (Faerber et al.,2007). New investigations indicate that alongside its anti-emeticeffects, tropisetron possesses notable immunomodulatory prop-erties (Fiebich et al., 2004a,b; Muller et al., 2006). In this regard, wehave recently shown that tropisetron and the congener granise-tron suppressed significantly the elevated pro-inflammatory cy-tokines tumor necrosis factor-alpha (TNF-) and interleukin-1(IL-1), neutrophil infiltration and lipid peroxidation in a ratmodel of colitis (Fakhfouri et al., 2010; Mousavizadeh et al.,2009). Moreover, we found that pretreatment with tropisetronsignificantly improved neurological deficits and diminished leu-kocyte transmigration into the brain, TNF- level and brain in-farction in a murne model of embolic stroke. The most strikingfinding of this study was that the mentioned salutary propertieswere not 5-HT3 receptor-dependent as granisetron, another se-lective 5-HT3 receptor antagonist, failed to elicit protective effects(Rahimianet al., 2011). Inhumanmonocytes, lipopolysaccharide-stimulated secretions of both TNF- and IL-1 were dose-dependently inhibited by tropisetron (Fiebich et al., 2004a,b).Findings of a recent study provide new mechanistic insightsinto anti-inflammatory and immunosuppressive activities of tro-pisetron; Vega, et al. demonstrated that tropisetron abates thetranscriptional activities of nuclear factor of activated T cells(NFAT), nuclear factor-kappa B (NF-B) and activator protein-1(AP-1). They proposed that calcineurin,may contribute to this ef-fect (Vega et al., 2005).

    The calcium-activated serine/threonine phosphatase calci-neurin is a key factor of a plethora of cell signaling processes,particularly, in immune, neuronal and muscle cells (Medyoufet al., 2007; Sieber and Baumgrass, 2009).

    Although calcineurin is abundant in neurons, accountingfor more than 1% of the total protein (Klee et al., 1998), itsrole in gene expression had not been investigated until re-cently, when the expression of certain isoforms of the inositoltrisphosphate receptor (IP3R), the plasma membrane Ca-ATPases (PMCA), and the Na/Ca exchanger (NCX) in culturedneurons were shown to be regulated by this phosphatase.IP3R was found to undergo upregulation upon calcineurin ac-tivation while PMCA4 and NCX2 undergo downregulation(Genazzani et al., 1999; Graef et al., 1999; Guerini et al., 2000;Li et al., 2000).

    All the genes known so far to be regulated by calcineurin inneurons appear to be involved in calcium (Ca) homeostasis,thereby providing some kind of activity-dependent reorganiza-tion of Ca-signaling at the transcriptional level (Kramer et al.,2003). Interestingly, Kramer et al. showed for the first time thatcalcineurin controls the expression of multiple proteins thatare not directly involved in calcium homeostasis. Cannabinoidtype one (CB1) receptor is one of the most important G-proteincoupled receptors the expression of which is regulated by calci-neurin. They also indicated that classical calcineurin inhibitorssuchas cyclosporine or tacrolimus (FK506) upregulateCB1 recep-tor expression (Kramer et al., 2003). Regarding the fact that tropi-

    2 B R A I N R E S E A R Cet al., 2004), anxiolytic (Costall et al., 1990; Lecrubier et al., 1993)anti-inflammatory (Schneider et al., 2004; Seidel et al., 2004)Fig. 1 Effect of tropisetron on cell viability. Cell viability wasevaluated by MTT reduction. Tropisetron (tropi) at the dosetron and granisetron at the dose range of 1 nM10 M did notsignificantly affect cell viability after 4 DIV, while at two higherdoses progressively reducedviable cells (data shown for tropise-tron at 4 DIV; Fig. 1). On this basis, the concentrations 100 M10 mM of either drug were excluded from further experiments.

    2.2. Effect of tropisetron on calcineurin activity in CGNculture

    Following 4 days of exposure, tropisetron significantly dimin-ished calcineurin activity at concentrations 100 nM10 M ascompared with the K25 control. Tropisetron at 110 nM failedto influence calcineurin activity (p>0.05, Fig. 2). The congenergranisetron produced no effect at the given concentrations(1 nM10 M; p>0.05, Fig. 3). After 2 DIV, none of the adminis-tered concentrations of tropisetron or granisetron affected calci-neurin activity (data not shown).(p

  • Fig. 2 Effect of tropisetron treatment for 4 DIV on calcineurinphosphatase activity in primary cerebellar granule cell culture.Tropisetron (tropi) significantly reduced calcineurin activity at100 nM10M concentrations (p
  • any given 5-HT3 antagonists, we did not proceed to performwestern blotting for this set of groups.

    2.6. Effect of tropisetron on cAMP level in CGN culture

    to the corresponding dose in K25 control culture (Fig. 6). This re-sult indicates that tropisetron acts to increase CB1-mediated sig-naling, i.e. inhibition of AC. Since neither granisetron nortropisetron affected CB1 mRNA expression after 2 DIV, the cul-tures treated over this period were not further investigated forcAMP content.

    3. Discussion

    Results of the present study show for the first time that tropise-tron inhibits the activity of the phosphatase calcineurin. Along-side, tropisetron upregulates CB1 expression at both transcrip-tional and translational levels in cultured cerebellar granulecells (CGNs). This effect is accompanied by a decrease in theCB1 receptor secondarymessenger, cAMP, content. Intriguingly,treatment of CGNs with granisetron another selective 5-HT3receptor antagonist did not affect the expression of CB1 recep-tor or its functionality. Furthermore, granisetron failed to altercalcineurin phosphatase activity under the same condition.The recognition that we and others showed the absence of5-HT3 receptors in cerebral granule cells (Kilpatrick et al., 1978;Maricq et al., 1991) together with the inability of congener gran-isetron to influence the mentioned pharmacological featuresimply that these effects of tropisetronwould occur independent

    Table 1 The effect of different treatments on mRNAexpression of CB1 receptor in CGN culture.

    Group CB1 expressionratio

    Result on mRNAexpression

    K25 1 No changeTropisetron 1 M 3.621.57 a Up-regulationTropisetron 10 M 5.8401.59 a Up-regulationGranisetron 1 M 0.8521.64 No changeGranisetron 10 M 0.9111.32 No change

    a p

  • plex formed between malachite green, molybdate and free

    H 1Although the exact mechanisms involved in the regulationof CB1 receptor expression are not yet fully understood, newfindings delineate that the alteration of intracellular Ca levelvia activation of intracellular cascades leads to changes in CB1receptor expression. Vallano et al. showed that the depolarizedcondition (medium containing 25mmol KCl) and the ensuinginward Ca down-regulate CB1 receptors and CB1-mediated sig-naling in CGNs. They found that nefidipine, an L-type Ca chan-nel blocker, up-regulated CB1 receptor in CGNs (Vallano et al.,2006). In another interesting research study, calcineurin wasshown to regulate the genes which are not solely engaged inCa homostasis; in this respect, calcineurin was implicated forthe first time in the expression of CB1 receptor. Their results in-dicate that calcineurin inhibitors such as tacrolimus (FK506)upregulate CB1 receptor expression (Kramer et al., 2003). In linewith this evidence, our data show that tropisetron potently in-hibits calcineurin activity in CGNs at 100 nM10 M. It is, there-fore, plausible that the observed CB1 receptor upregulation is aconsequence of inhibition of calcineurin by tropisetron.

    To determine whether tropisetron-induced upregulation ofCB1 in granule neurons also affects the receptor signalingmedi-ated by adenylyl cyclase, the effect of a potent synthetic canna-binoid receptor agonist, WIN 55212-2, was assessed onforskolin-stimulated cAMPgeneration inCGNs treatedwith tro-pisetron. For this reason, CGNs received for 4 DIV 10 M tropise-tron which had exerted the highest effect on CB1 receptorupregulation. In tropisetron treated CGNs, WIN 55212-2 signifi-cantly decreased the intracellular cAMP content in comparisonto CGNs in theK25 control. This implies that upregulation of CB1receptors is associated with activation of CB1 signalingpathway.

    Activation of cannabinoid signaling might take part inmany pharmacological aspects of tropisetron including anti-inflammatory, neuroprotective, anxiolytic, and analgesic ef-fects. Classically, tropisetron is the antagonist of 5-HT3 recep-tors which belong to the ligand-gated ion channel family(Faerber et al., 2007). Mechanisms other than antagonism of5-HT3 receptors could be intriguing from a pharmacologicviewpoint and could be a basis for new drug development.

    In summary, tropisetron-induced calcineurin inhibitionmight underlie the alteration seen in CB1 receptor expression.However, more investigations can further unravel the mecha-nisms involved in such novel pharmacological aspect of tropi-setron. In addition, studies should be designed to clarifywhether tropisetron can exert such traits in vivo. Finally, as tro-pisetron and cannabinoids share several pharmacological prop-erties, combination therapy of tropisetron and cannabinoidscould be considered to curtail adverse effects of cannabinoids,in particular psychotropic, to exploit possible additive or syner-gistic effects, and to minimize the doses of each medication.

    Although tropisetron targets calcineurin, a pivotal enzymein activating transcriptional factors responsible for immune/inflammatory axis regulation, it is yet to be delineated whethertropisetron directly inhibits calcineurin or else it acts via calci-neurin interacting molecules such as immunophilins. The factthat tropisetron inhibits calcineurin activity could open an ave-nue to develop novel calcineurin inhibitors with tolerable sideeffects. So far, no immunosuppressive properties have been

    B R A I N R E S E A R Creported by tropisetron in humans. It might be because theanti-emetic doses administered do not elicit such effect or it isphosphate released. CGNs were detached from plates by scrap-ing, rinsed in ice-cold tris buffer solution (TBS) and counted.Five million cells were lysed in 1ml of the provided lysis bufferconcurrently used with chemotherapeutics which per se pos-sess immunosuppressive action (Vega et al., 2005).

    4. Experimental procedures

    4.1. Cell culture

    Cerebellar granule cellswere dissociated from the cerebella o...


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