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Research Report

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

Reza Rahimiana, Ahmad Reza Dehpoura, Gohar Fakhfourib,Mohammad Reza Khorramizadehc, Jean-Eric Ghiad, Mohammad Seyedabadia,Antonio Caldarellie, Kazem Mousavizadehf, Mehdi Forouzandehg, Shahram Ejtemaei Mehra,⁎aDepartment of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, IranbDepartment of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, IrancDepartment of Medical Biotechnology, School of Advanced Medical Technologies, Tehran University of Medical Sciences, Tehran, IrandDepartment of Immunology & Internal Medicine Section of Gastroenterology, Faculty of Medicine, University of Manitoba, Manitoba,Winnipeg, CanadaeDipartimento di Scienze Chimiche Alimentari Farmaceutiche e Farmacologiche, Università del Piemonte Orientale A. Avogadro, Novara, ItalyfCellular and Molecular Research Center, Tehran University of Medical Sciences, Tehran, IrangDepartment of Medical Biotechnology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran

A R T I C L E I N F O

⁎ Corresponding author at: P.O. Box: 13145-78E-mail address: ejtemam@gmail.com (S.EAbbreviations: CB1, cannabinoid type one;

0006-8993/$ – see front matter © 2011 Elseviedoi:10.1016/j.brainres.2011.08.050

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 immunophilin–calcineurinpathway. 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 congenergranisetron at 1 nM–10 μM concentrations. Moreover, cannabinoid CB1 receptor expressionat 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.Our 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.

Keywords:TropisetronCannabinoid receptorCerebellar granule neuronsCalcineurin

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

r B.V. All rights reserved.

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1. Introduction properties and taking into account the ability of calcineurin to

Fig. 1 – Effect of tropisetron on cell viability. Cell viability wasevaluated by MTT reduction. Tropisetron (tropi) at the doserange of 1 nM–10 μM did not affect cell viability after 4 DIV,while at 100 μM–1 mM significantly reduced viable cells(p<0.05). 100 corresponds to the MTT value for K25 control.* p<0.05 vs K25 control.

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-α) and interleukin-1β(IL-1β), 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-α 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-α and IL-1β 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-κB) 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-setron and CB1 receptor agonists share various pharmacologicaleffects including anti-emetic (Scuderi, 2001) analgesic (Rieringet al., 2004), anxiolytic (Costall et al., 1990; Lecrubier et al., 1993)anti-inflammatory (Schneider et al., 2004; Seidel et al., 2004)

control 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 nM–1 mM) or granisetron (1 nM–1 mM)on cell viability were assessed usingMTTmethod. Both tropise-tron and granisetron at the dose range of 1 nM–10 μM 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 μM–10 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 nM–10 μM ascompared with the K25 control. Tropisetron at 1–10 nM failedto influence calcineurin activity (p>0.05, Fig. 2). The congenergranisetron produced no effect at the given concentrations(1 nM–10 μM; 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).

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 nM–10μM concentrations (p<0.05), while producing noeffect at 1–10 nM. * p<0.05 vs K25 control.

Fig. 4 – A. Expression of 5-HT3 receptors in cerebellar granuleneurons. cDNAobtained fromCGNswasassessed for presenceof 5-HT3. Rat whole brain and intestine served as positivecontrols. The band (172 bp) corresponding to the functionalsubtype 5-HT3A (5-TH3) is not detectable in CGNs. β2

Macroglobulin (B2M) was used as the house keeping gene.B. Effect of tropisetron treatment for 4 DIV on CB1 receptorprotein expression in primary cerebellar granule cell culture.Tropisetron (tropi) significantly upregulated CB1 receptorprotein expression at 100 nM–10 μM concentrations (p<0.05).Granisetron (grani) produced no effect at 1–10 μM. The imagesshow representative Western blots of CB1 receptor proteinlevels (n=5 independent cultures), and the bar graph showsquantitative densitometric analysis of CB1 receptor. Data arepresented as OD after normalization with correspondingβ-actin level. * p<0.05 vsK25 control. × p<0.05 vs tropi 100 nM.

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2.3. 5-HT3 receptor expression in CGN culture

To assess the presence of 5-HT3 receptors in CGNs, oligonucleo-tide primers specifying a product of 172 bp corresponding to5-HT3A mRNAwere used in a standard PCR assay. As illustratedin Fig. 4A, the ratwhole brain and intestine yielded amplicons of172 bp, while in CGNs samples 5-HT3A PCR amplification failedto produce any detectable products, suggesting the absence ofthis receptor in cultured CGNs. The house keeping gene usedwas β2-Macroglobulin.

2.4. Effect of tropisetron onmRNA expression of CB1 receptorin CGN culture

The result of data analysis with REST 2008 indicated that CB1

mRNA was up-regulated in cultures treated with tropisetron for4 DIV as compared with K25 control. The fold change in expres-sion ratio of CB1 receptor in tropisetron-treated versus non-treated group was 3.62 and 5.84 for 1 μM and 10 μM tropisetron

Fig. 3 – Effect of granisetron treatment for 4 DIV on calcineurinphosphatase activity in primary cerebellar granule cell culture.Granisetron (grani) produced no significant effect oncalcineurin activity at 10 nM–10 μM concentrations (p>0.05).* p<0.05 vs K25 control.

respectively (p<0.001, Table 1). In 4 DIV granisetron treatedgroup (1–10 μM), however, no change was seen in expressionratio of CB1 mRNA (p>0.05, Table 1). After 2 DIV, tropisetron(1–10 μM) and granisetron (1–10 μM) did not alter CB1 mRNA ex-pression (data not shown).

2.5. Effect of tropisetron on protein expression of CB1 receptorin CGN culture

After 4 DIV, tropisetron caused a significant over-expressionof CB1 receptor protein levels in CGNs at all three concentra-tions used (100 nM–10 μM) in comparison with K25 control.Over-expression of the CB1 receptor protein was significantlyhigher in 10 μM and 1 μM tropisetron-treated cultures com-pared with those receiving tropisetron at 100 nM concentra-tion (p<0.05, Fig. 4B). Granisetron, however, did not influencethe CB1 receptor expression (p>0.05, Fig. 4B). As no alterationin CB1 receptor was observed at mRNA level after 2 DIV with

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 μM 3.62±1.57 a Up-regulationTropisetron 10 μM 5.840±1.59 a Up-regulationGranisetron 1 μM 0.852±1.64 No changeGranisetron 10 μM 0.911±1.32 No change

a p<0.001 compared to K25 culture group. Treatments were givenfor 4 DIV.

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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

Due to coupling to inhibitory G-proteins (Gi/o), CB1 receptor,when acutely stimulated, conventionally causes an inhibitionof cAMP production through inhibition of adenylate cyclase.This feature was exploited to examine whether the increaseby tropisetron of CB1 protein expression and mRNA levels alsolead to an alteration in CB1-mediated signal transduction. Tro-pisetron at 10 μM concentration was used as it caused a greaterCB1 mRNA and protein over-expression. Application of forsko-lin (1.0 μM) 10min before harvesting granule cells resulted in adrastic rise in cAMP levels in both K25 control and cultures trea-ted for 4 DIV with 10 μM tropisetron (p<0.05, Fig. 5). No differ-ence was seen between K25 control and tropisetron treatmentin the forskolin-stimulated increase of cAMP, thus demonstrat-ing that the Adenylate cyclase (AC) production of cAMP wasequally intact in both groups. Acute exposure with WIN 55212-2, a potent cannabinoid receptor agonist, at 100 nM for 10min be-fore an additional 10min treatment with forskolin significantlydiminished cAMP concentrations in two mentioned cultures.Consistent with the results from western blotting and real timePCR assays, in tropisetron-treated cultures WIN 55212-2 causeda significantlymore reduction in cAMP concentrations compared

Fig. 5 – Effect of tropisetron treatment for 4 DIV on cAMP levelin primary cerebellar granule cell culture. Application offorskolin (FSK; 1.0μM) 10 min before harvesting granule cellssignificantly increased cAMP levels in all K25 control cultureand cultures treated for 4 DIV with either 10 μM tropisetron(tropi) or 10 μM granisetron (grani). No significant differencewas seen among FSK effects on three mentioned cultures.* p<0.05 vs K25 control, × p<0.05 vs tropi, + p<0.05 vs grani.

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-HT3

receptor 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 independentof 5-HT3 receptor, itsmain site of action known so far. Results ofthis study parallel our recent investigation indicating tropise-tron, but not granisetron, protects against embolic model ofstroke in a 5-HT3 independentmanner. Our findings also justifythe inability of granisetron to influence IL2 production, a cyto-kine regulated by calcineurin, in Jurkat cell line (Rahimianet al., 2011; Vega et al., 2005).

Fig. 6 – Effect of WIN 55212-2 on cAMP level in primarycerebellar granule cell culture treated for 4 DIV withtropisetron. Acute exposure with WIN 55212-2 (WIN) at200 nM for 10min before an additional 10 min treatment withforskolin (FSK) significantly diminished cAMP concentrationsin K25 control, tropisetron-treated (tropi) andgranisetron-treated (grani) cultures. In tropi cultures, WINcaused a more significant reduction in cAMP concentrationscompared to the corresponding dose in K25 control. cAMPcontent did not differ between K25 control and grani culturesafter incubation withWIN. Values are expressed aspercentage of FSK-stimulated cAMP, set at 100%(n=5 independent cultures). * p<0.05 vs respective control,× p<0.05 vs WIN.

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Although 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 CB1

receptor 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 nM–10 μM. 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 μM 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 CB1

receptors 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 beenreported by tropisetron in humans. It might be because theanti-emetic doses administered do not elicit such effect or it is

concurrently 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 of 7-day-oldWistar rat pups as described (Kramer et al., 2003). Brief-ly, the cerebella were removed, rinsed in HBSS-BSA, minced,digested with 0.025% trypsin and incubated at 37 °C for 15 min.To stop the digestion, DMEM containing 10% fetal calf serumwas added; then a single cell suspension was obtained throughpipetting up and down the sedimented tissue. Following centri-fugation, cells were counted using trypan blue exclusion test ina Burker chamber. This method is based on the ability of viablecells to exclude trypan blue due to their intact cell membranes,leaving them unstained while nonviable cells take up the dye.Cells at 3.2×105/cm2 were seeded in DMEM Hepes modificationsupplemented with 10% fetal calf serum, 100 μg/ml pyruvate,and 100 μg/ml gentamicin on Polysyrene 12-well tissue cultureplates (CELLSTAR) coated with poly-L-lysine. After 24 h, 10 μMcytosine arabinofuranoside was added to inhibit the growth ofnon-neuronal cells. All pharmacological interventions beganat this time in the culture medium containing 25mM KCl (K25)which causes depolarization of granule cells and consequentlyactivates gene expression machinery. Moreover, the viabilityof granule neurons is higher in this milieu and therefore pro-vides the possibility to maintain cells for several days. Indeed,25 mMKCl is required in themedium for CGN cultures tomain-tain adequate calcineurin activity and cell maturation and de-polarization (Kramer et al., 2003; Vallano et al., 2006). Culturesreceived simple medium (K25 control) or medium containingtropisetron or granisetron for 2 or 4 days in vitro (DIV).

4.2. Cell viability (MTT) assay

Cellular viabilitywas assessedusing 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay based on the abil-ity of living cells to reduce a yellow tetrazolium-based compoundto a formazan product. CGNs plated at 3.2×105/cm2werewashedwith Locke solution (134mM NaCl, 25mM KCl, 4 mM NaHCO3,10mM HEPES·NaOH, 2.3 mM CaCl2, 1 mM MgCl2, 5 mM glucose)and incubated with MTT in Locke (0.25mg/ml) at 37 °C for 1 h.Then a 0.1 M HCl in isopropanol was added to dissolve the insol-uble purple formazan product into a colored solution. The absor-bance of this colored solution was quantified by measuring at570 nm by a spectrophotometer (Vallano et al., 2006).

4.3. Calcineurin activity assay

Phosphatase calcineurin activity was assessed in CGNs using acolorimetric assay kit based on quantification of the green com-plex formed between malachite green, molybdate and freephosphate 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 bufferand centrifuged at 150,000×g at 4 °C for 45min, and the super-natant was stored at −70 °C until analysis. Prior to calcineurin

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activity assay, free phosphate and nucleotides were removedfromcellular extract by gel filtration. To ensure the complete re-moval of phosphate,malachite green reagentwas added,whichwould turn the color into green inpresence of phosphate. A spe-cific calcineurin substrate was added to phosphate-free cellularextracts and after appropriate incubation period, malachitegreen reagent was applied.

The rapid green color formation from the reaction wasmea-suredona spectrophotometer. Thedeveloped colorwaspropor-tional to the calcineurin phosphotase activity of samples.Absorbance values, read at 620 nm, were transformed to per-centage and values pertaining to K25 control were considered100%. For every single treatment, five independent cultureswere assessed and each sample was run in triplicate.

4.4. Polymerase chain reaction

PCR was performed for investigating the presence of 5-HT3 re-ceptors in CGNs. Rat whole brain and intestine cDNA servedas positive controls. 5-HT3A forward primer was 5′-GGCCCATA-GACCCCCAGCCA-3′, and reverse primer 5′-CAAGAGACCGCG-CACAGCCA-3′. Prior to PCR, the samples were incubated at94 °C for 3 min. PCR was performed in Biometra thermocycler(model T3000) for 30 cycles as follows: 95 °C for 20 s, 60 °C for20 s, 72 °C for 20 s. Afterward the samples were heated to 72 °Cfor 5 min. The amplicons were resolved on 1% polyacrylamidegel and photographed under UV illumination.

4.5. Real-time PCR

4.5.1. RNA extractionTotal RNA was isolated from primary granule neurons usingHigh Pure RNA Isolation Kit following themanufacturer's direc-tions. Due to the integrated DNase digestion step, contamina-tion of the isolated RNA with residual genomic DNA wasmostly avoided. The concentration and purity of RNA wereassessed spectrophotometrically using nucleotide absorptionat 260 nM and ratio at 260/280 nm (nucleotide/protein), respec-tively. The isolated RNA had absorbance 260/280 ratios equalor greater than 1.8.

4.5.2. Reverse transcriptionRNAwas used in RT-reaction to produce cDNA using Transcrip-tor Reverse Transcriptase kit for use as template in PCR. 1 μg oftotal RNA in 13 μl of PCR grade water containing oligo (dT)15primer (5 μM) was heated to 65 °C for 10min then cooled onice to ensure denaturation of RNA secondary structures. 7 μl ofa mixture containing RNase inhibitor (20 U), RT buffer (50 mMTris/HCl, 30 mM KCl, 8 mM MgCl2, pH 8.5), the four dNTPs(1 mM each), and reverse transcriptase (10 U) was added toeach RNA sample (final volume, 20 μl). The RT reaction was ini-tiated by incubation for 30 min at 55 °C to promote synthesis ofcDNA, and terminated by heating to 85 °C for 5 min.

4.5.3. Real time polymerase chain reactionReal timequantitativeRT-PCR (qRT-PCR) analysiswas carried outas the most sensitive method applied so far for quantification ofdifferences in mRNAs. The main feature of this technique is useof a fluorescent dye, here SYBR Green, which emits fluorescenceuponbinding thedouble strandedDNA.Therefore, theamountof

fluorescence detected during the extension phase is proportionalto the amount of amplicons (Vallano et al., 2006). In order to am-plify cDNA, the polymerase chain reactionwas performed in Ste-pOnePlus real time PCR System (Applied Biosystems, CA, USA)with β-actin as the internal standard. The primer sequences forthe gene and internal standard were as follows: β-actin forwardprimer 5′-GGAAATCGTGCGTGACATTAAA-3′, β-actin reverseprimer 5′-TGCGGCAGTGGCCATC-3′, CB1 forward primer 5′-ATGAAGTCGATCCTAGATGGCCTTG-3′, and CB1 reverse primer5′-GTTCTCCCCACACTGGATG-3′. Total volume of the reactionwas 12.5 μl which contained 10 pmol of each primer, 6 μl ofPower SYBR Green PCR Master Mix 2X and 1.2 μl of template.The holding stage (95 °C for 5 min) was followed by the cyclingstage (denaturation 10 s at 95 °C, combined annealing/extension30 s at 60 °C) and the number of cycles was 40.

4.6. Western blotting

Granule cells were exposed to tropisetron (10 nM–10 μM) orgranisetron (1–10 μM) as indicated. Granule cells were washedin phosphate buffered saline (PBS), collected in a microfugetube, lysed in ice-cold Nonidet P-40 buffer solution [20 mMTris HCl (pH 8), 137 mM NaCl, 10% glycerol, 1% Nonidet P-40,2 mM EDTA, 1 mM sodium orthovanadate, 50 mM sodiumfluoride, 1 mM phenylmethylsulfonyl fluoride, 2 μg/ml aproti-nin, 10 μg/ml leupeptin, 10 μg/ml antipain, and 1 μg/ml pep-statin A] and centrifuged at 12,000×g at 4 °C for 20 min(Zhang et al., 2007). Supernatants were collected and total pro-tein amounts determined by the Bradford assay, using bovineserum albumin as a standard. 20 μg of proteins was boiled inSDS-mercaptoethanol sample buffer and electrophoresed in10% SDS/polyacrylamide gels. Separated proteins were trans-ferred onto PVDF membranes at 120 V, for 1.15 h and blockedin TBS solution containing 0.1% Tween 20 and 5% non-fatdry milk at room temperature for 1 h. Membranes were incu-bated with rabbit anti-CB1 receptor antibody raised against Cterminal amino acids 461–472 of Cannabinoid Receptor I(1:200) overnight at 4 °C. Blots were then washed 3 times,probed with horseradish peroxidase (HRP)-conjugated goatanti-rabbit antibody (1:3000) for 1.15 h and developed usingan enhanced chemiluminescence system (ECL). The bandsrepresenting CB1 receptor protein (60 kDa) were quantifiedby densitometry using GeneTools (Syngene, Cambridge, UK)and normalized to β-actin band intensity.

4.7. cAMP assay

CGNs at a density of 3.2×10−5/cm2 were grown with or withouttropisetron (10 μM) for 4 DIV. Culture medium was replacedwith simple medium or that containing the cannabinoid recep-tor agonist, WIN (100 nM) for 10 min. In another set of experi-ments, 10 min following the addition of WIN, forskolin (FSK,1 μM) was also added for further 10 min. Cells were washed,lysed in 0.1 M HCl (500 μl/well), and centrifuged at 600×g. Super-natants were collected and assayed for cAMP using Direct CyclicAMPEnzyme ImmunoassayKit, according to themanufacturer'sinstructions. In brief, samples or standards (0–20 pmol/ml) wereloaded in wells pre-coated with an antibody mounted againstcAMP. Afterwards, cAMP conjugate and cAMP antibody wereadded and the reaction was shaken at room template for 2 h.

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After threewashes, the chromogenic substrate p-Nppwas addedand color allowed to develop for 1 h. Optical density (OD) wasread at 405 nm immediately after addition of stop solution. Theintensity of the yellow color generatedwas inversely proportion-al to the concentration of cAMP in either standards or samples.

5. Experimental procedures

Tropisetron (Sigma, St. Louis, MO, USA); granisetron (Sigma,St. Louis, MO, USA); calcineurin cellular activity assay kit(Cabiochem, Darmstadt, Germany); high Pure RNA IsolationKit (Roche Applied Science, Mannheim, Germany); transcrip-tor Reverse Transcriptase kit (Roche Applied Science, Mann-heim, Germany); Direct Cyclic AMP Enzyme ImmunoassayKit (Assay Designs, MI, USA); DMEM (Sigma, St. Louis, MO,USA); bovine serum albumin (BSA, Sigma, St. Louis, MO,USA); fetal calf serum (FCS, Biosera, Sussex, UK); poly-L-lysine(Sigma, St. Louis, MO, USA); PCR Master Mix 2X (applied bio-systems, CA, USA); PVDF membranes (Millipore Corporation,Bedford, MA, USA); rabbit anti-CB1 receptor antibody raisedagainst C terminal amino acids 461–472 of Cannabinoid Re-ceptor I (AbCam); horseradish peroxidase-conjugated (HRP)-conjugated goat anti-rabbit antibody (1:3000; Cell SignalingTechnology, Beverly, MA, UK); enhanced chemiluminescencesystem (ECL; Amersham Biosciences, Buckinghamshire, UK).

5.1. Statistics

For MTT assay, calcineurin activity, western blotting and cAMPassay, statistical analysis was performed using One-wayANOVA followed by Tukey post-hoc test for multiple compari-sons. Results were presented as mean±SEM and a p value lessthan or equal to 0.05 considered significant. For analysis of realtime PCR data, the relative expression software tool (REST) 2008(Corbett researchPty,V 2.0.7)was employed (Mohammadi-Faraniet al., 2010). The fold change expression ratio of genes in this soft-ware was calculated using the 2−ΔΔCt (relative quantification)method, where Ct is the cycle at which the fluorescence from asample crosses the threshold. The principles of calculations arepreviously described (Livak and Schmittgen, 2001; Pfaffl et al.,2002). In brief, REST uses Pair Wise Fixed Reallocation Randomi-zation Test, which is more flexible than non-parametric testsbased on ranks and do not suffer a reduction in power relativeto parametric tests. REST, developed for quantification ofmRNA, exhibits suitable reliability as well reproducibility in indi-vidual runs, confirmed by high accuracy and low variation inde-pendent of huge template concentration variations.

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