European Journal of Pharmacology 650 (2011) 28–33

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European Journal of Pharmacology

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Molecular and Cellular Pharmacology

Intracellular cAMP assay and Eu-GTP-γS binding studies of chimeric opioidpeptide YFa

Krishan Kumar a,b,c, Sambuddha Kumar a, Raj Kumar Kurupati a, Mahesh Kumar Seth a,d, Anita Mohan b,M Ejaz Hussain d, Santosh Pasha a,⁎a Institute of Genomics and Integrative Biology (CSIR), Mall Road, Delhi, Indiab University School of Basic and Applied Sciences, GGS I P University, Kashmere Gate, Delhi, Indiac Department of Chemistry, Motilal Nehru College, University of Delhi, Delhi, Indiad Department of Biosciences, Jamia Millia Islamia, New Delhi, India

⁎ Corresponding author. Peptide Synthesis LaboratoIntegrative Biology, CSIR, Mall Road, Delhi 110007, Infax: +91 11 27667471.

E-mail address: spasha@igib.res.in (S. Pasha).

0014-2999/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.ejphar.2010.09.026

a b s t r a c t

a r t i c l e i n f o

Article history:Received 6 January 2010Received in revised form 26 July 2010Accepted 14 September 2010Available online 29 September 2010

Keywords:Cyclic adenosine monophosphateAntinociceptionAdenylyl cyclaseTolerance

In our previous studies chimeric peptide of Met-enkephalin and FMRFa, YGGFMKKKFMRFamide (YFa),demonstrated concentration dependent κ– and μ–opioid receptor mediated antinociception withouttolerance development. To gain further insight of the observed behavior of YFa, the present study wasundertaken. The effect of chimeric peptide on forskolin-stimulated cAMP formation under acute and chronictreatment and stimulation of Eu-GTP-γS binding in CHO cells stably expressing κ– and μ–opioid receptors wasassessed. YFa showed concentration dependent inhibition of forskolin-stimulated cAMP in both hKOR andhMOR-CHO cells; however, the inhibition at 1 nM was significantly higher in hKOR cells and comparable toDynA (1–13) than that shown at 20 nM in hMOR cells. Chronic treatment of YFa, similar to DynA (1–13), didnot show significant change in forskolin-stimulated cAMP level in both hKOR and hMOR cells. However,chronic treatment of morphine and DAMGO showed an increase in forskolin-stimulated cAMP level in hMOR-CHO cells indicating superactivation of adenylyl cyclase. Eu-GTP-γS binding studies of YFa showed aconcentration dependent adherent binding with κ– and μ–opioid receptors; however, the latter demonstratedsignificant binding at higher concentration. Thus the study indicates the chimeric opioid peptide YFa as a potentκ- receptor specific antinociceptivemoiety, showingno tolerance andhencemay serve as a lead inunderstandingthe mechanism of tolerance development, antinociception and its modulation.

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1. Introduction

Opioids are widely used as analgesic in medicine. They exert theiraction through various known opioid receptors which have beenclassified pharmacologically into three subtypes: μ–, δ– and κ–receptors (Loh and Smith, 1990). Opioid receptors are G-protein-coupled receptors (Raynor et al., 1994) that are activated by opioidpeptides to mediate their action in the central nervous system (CNS)and the peripheral nervous system (Binder et al., 2001; Newman et al.,2002). The signaling involves the activation of specific heterotrimericG-protein and subsequent dissociation of α and βγ subunits. Thesesubunits serve as the activators or inhibitors of several effector systemsincluding adenylyl cyclases, phospholipases and ion channels (Pierceand Lefkowitz, 2001). Apparently, the cellular adaptations detected inacute opioid treatment lead to the inhibition of cAMP activity.However prolonged agonist treatment not only blunts these cellular

responses but also compensates the increase in intracellular cAMP(Law et al., 1982; Pineyro and Archer-Lahlou, 2007; Sharma et al.,1977). This compensatory adenylyl cyclase activation phenomenonhas been postulated to be responsible for the development of drugtolerance and dependence (Koob and Bloom, 1988). Receptordesensitization, phosphorylation, Src phosphorylation and endocyto-sis are molecular mechanisms contributing to the development oftolerance and dependence (Finn and Whistler, 2001; Xu et al., 2007;Waldhoer et al., 2004; Zhang et al., 2009) and are also implicated in theswitching of opioid receptor fromGi/Go to Gs during chronic treatment(Szucs et al., 2004), nonetheless the exact molecular mechanisms stillremain elusive.

Methionine-enkephalin-Arg6-Phe7 (MERF) peptide that belongs tothe opioid family (Inturrisi et al., 1980) is comprehensively distributed inthe CNS of differentmammals (Majane et al., 1983). Conversely, peptidesof the NPFF/FMRFa family potently antagonize morphine-inducedsupraspinal analgesia (Tanget al., 1984) andmay functionas endogenous‘anti-opioid’ agents (Galina and Kastin, 1986). NPFF has also beenperceived to exhibit opioid effects along with a role in tolerance. Theintriguing relationship between opioids and anti-opioids is attributed toFMRF amino acid sequence contained at C-terminal of MERF. Based on

Fig. 1. (A)Western blots of μ– and κ–opioid receptor proteins in hMOR-CHO and hKOR-CHO cells. (B) The relative intensity of μ– and κ–opioid receptor bands against loadingcontrol (C) The graphical representation shows the relative integrated densitometryvalues (IDVs) quantified and normalized by that of the GAPDH signal Alpha Ease FCsoftware.

29K. Kumar et al. / European Journal of Pharmacology 650 (2011) 28–33

MERF, a chimeric peptide ‘YFa (YGGFMKKKFMRFamide)’, of met-enkephalin and FMRFa was designed to determine the role ofendogenous amphiactive sequences like MERF in analgesia and itsmodulation (Gupta et al., 1999).

Our investigations on YFa demonstrated that IP administration ofthe peptide induced nalaxone-reversible antinociception suggestingthe role of opioid receptors in mediation of its antinociceptive effects.Furthermore, YFa potentiated morphine induced antinociception andattenuated the development of tolerance to morphine analgesia,indicating its possible role in pain modulation (Gupta et al., 1999).Moreover, mRNA expression studies revealed that YFa producedκ-receptor specific antinociception with no tolerance (Vats et al.,2008) and also induced cross tolerance to 20 mg/kg morphine anal-gesia after 4 days pretreatment with 80 mg/kg YFa (Gupta et al.,2008). The present study was designed to understand the cellularadaptations at second messenger level e.g. cAMP inhibition andGTP binding of YFa on interaction with opioid receptors. Therefore,dose dependent Eu-GTP-γS binding assay and forskolin-stimulatedcyclic AMP inhibition studies of YFa were designed and performedin hKOR-CHO and hMOR-CHO cell lines stably expressing κ– andμ–opioid receptors, respectively.

2. Materials and methods

2.1. Peptide synthesis

Peptides, YFa, and dynorphin A (Tyr1-Gly2-Gly3-Phe4-Leu5-Arg6-Arg7-Ile8-Arg9-Pro10-Lys11-Leu12-Lys13) (DynA(1–13)) were synthe-sized by the solid-phase method on an ACT-90 peptide synthesizer(Advanced ChemTech, Louisville, KY) using the standard chemistry of9-flourenylmethoxycarbonyl amino acids (Novabiochem, Laufelfigen,Switzerland) and 1-hydroxybenzotriazole/diisopropylcarbodiimideactivation method on Rink amide-MBHA and Wang resin, respective-ly. The peptides were purified by RP-C18 column (mBondapakTM10 mm, 7.8 mm_ 300 mm, Waters, USA) on semipreparative reversephase HPLC (Waters 600, USA)with a 40 min linear gradient from 10%to 90% acetonitrile containing 0.05% trifluoroacetic acid in water. Themass analysis of the peptides was done in linear positive ion mode byMALDI-TOF/TOF (Bruker Daltonics Flex Analysis, Germany) with 2,5-dihydroxybenzoic acid as the matrix. The peptide sequence wasconfirmed by automated peptide sequencing (Procise 491, AppliedBiosystems, USA).

2.2. Chemicals

All the chemicals including DAMGO, IBMX, Naloxonazine, nor-Binaltorphimine (nor-BNI) and Forskolin were purchased from Sigma(St. Louis MO). Morphine was procured from AIIMS, New Delhi, India.All the peptides were dissolved in Milli-Q water.

2.3. Cell culture and membrane preparation

The recombinant CHO cells (hMOR-CHO and hKOR-CHO), stablytransfected with human μ–opioid receptor (hMOR) and κ-opioidreceptor (hKOR) DNAwere the kind gift from Prof. R.B. Rothman, NIH,Maryland, USA. The cells were grown in plastic flasks in DMEM/HAMSF-12 (50/50) (hMOR-CHO) and DMEM (hKOR-CHO)medium contain-ing 10% FBS, 100 units/ml penicillin,100 mg/ml streptomycin, andG418(0.20–0.25 mg/ml) under 95% air/5% CO2 at 37 °C. Tranformantcells, expressing target GPCR in appropriate medium, were grown tosubconfluency level. The cells were collected in phosphate bufferedsaline containing 2.7 mM EDTA. The cells were homogenized inhomogenizing buffer (10 mM NaHCO3, 5 mM EDTA, 0.5 mM PMSF,20 μg/ml eupeptic, 10 μg/ml pepstatin, 8 μg/ml E-64, pH 7.3) using apolytron homogenizer at 40 °C. The cell homogenate was spun at700×g for 10 min and the supernatant was collected and was spun at

100,000×g for 60 min to obtain the pellet, which was suspended inbinding buffer and used for Eu-GTP-γS binding assays. Westernblotting of the suspension was performed to check the expression ofκ–and μ–receptors in hKOR and hMOR cells, respectively (Fig. 1). Forthe drug pretreatment experiments, cells were incubated in freshmedium with increasing ligand concentrations (from 0.5 nM to250 nM) of YFa, DynA (1–13), Morphine, DAMGO, Naloxonazine,nor-BNI and forskolin for 20 h.

2.4. Western blotting

The expression of κ– and μ–opioid receptors in CHO cell lineswas evaluated by western blotting. Fifty microgram of total protein(3 μg/μl; Lowery assay) was resolved by electrophoresis, transferredto nitrocellulose membranes and blocked by incubation with a 5%skimmed milk solution in 1X Tris Buffered Saline Tween 20(TBST)(0.1 M Tris–HCl, pH 7.4 containing 0.9% NaCl and 0.1% Tween-20) for1 h at ambient temperature (25 °C–28 °C). The membranes wereincubated with the rabbit polyclonal anti-MOR1 antibody and anti-KOR1 antibody (Sigma, St. Louis, MO) and β-actin (Santa cruz) at adilution of 1:1000 in 1% skimmed milk in 1X TBST with constantshaking at 4 °C overnight. The membranes were then incubated for1.5 h at room temperature with secondary antibody (goat anti rabbitIgG-ALP) at a dilution of 1:2000. Subsequent detection was done with5-Bromo-4-Chloro-3′-IndolylPhosphate p-Toluidine-Nitro-Blue Tet-razolium Chloride (BCIP-NBT). GAPDHwas used as the loading controlin all cases (Fig. 1).

2.5. Cyclic AMP assay

Intracellular cAMP assay is a powerful technique to functionallyscreen new ligands for their specificity towards opioid receptors(Gupta et al., 2006). Cyclic AMP level was assessed as per Hit-HunterscAMP for Adherent cells EFC Fluorescent Detection kit (Amersham 90-0001-02 384 well format). Forskolin was used as a positive control–cAMP stimulant. For acute treatment experiment, the cells wereincubated with agonist and controls for 30 min and for the chronicexperiment the incubation time was 20 h. The cells, plated in 100 μl/well (20,000 cells in each well) in a 96-well were incubated overnightat 37 °C in an atmosphere of 5% CO2 and 95% air. The media wasaspirated gently and 80 μl/well of Hanks' buffer or Krebs Ringer buffercontaining 1 mM 3-isobutyl-1-methylxanthine (IBMX)-a photo

Fig. 2. % Inhibition of forskolin-stimulated cAMP formation in hKOR-CHO cells in acutetreatment of 30 m. Values represent mean±S.E.M. for three experiments performed intriplicate. IC50 values were calculated by one way ANOVA and data was significant with(P=0.003). When YFa was incubated with kappa specific antagonist nor-BNI itdemonstrated the reversibility in activity (i.e. 33% at 100 nM, P=0.003).

Table 1IC50 values of forskolin-stimulated cAMP formation in acute treatment of YFa andcontrols for kappa and mu receptor in hKOR and hMOR cells.

hKOR-CHO hMOR-CHO

Agonist IC50 Log(nM)*[n=14]

Agonist IC50 Log(nM)*[n=14]

DAMGO −8.013DynA(1–13) −7.990 Morphine −7.942YFa −7.826 YFa −7.413YFa+norBNI Not significant YFa+Naloxonazine Not significant

*P values for hKOR and hMOR cells were statistically significant; (P=0.003) and(P=0.001), respectively. The values represent mean±S.E.M. for three experimentsperformed in triplicate. IC50 values were calculated by one-way ANOVA and data werestatistically significant.

30 K. Kumar et al. / European Journal of Pharmacology 650 (2011) 28–33

diesterase inhibitor was dispensed. Each well was labeled as BKG,Standard, forskolin, agonist with forskolin and zero control. Plate wasincubated for 10 min at room temperature. Then in the first step,agonist/PBS was added and incubated for 30 min at 37 °C. In thesecond step, cells were treated with Std/Lysis/Ab or Lysis Ab inrespective wells and were incubated for 60 min at 37 °C. In the thirdstep, ED/substrate/ BKG solutions were added and incubated for60 min at 37 °C. In the 4th step, EA was added in each well andincubated for 1–3 h at room temperature. Finally, plate was read byFluoromax upon excitation at 530 nM and emission at 595 nM. Forconcentration–response curves, cells were treated with increasingligand concentrations from 0.5 nM to 250 nM. Two independentexperiments were conducted in triplicate with hKOR and hMOR cellsand the data were analyzed with Origin 7.1 to calculate IC50(concentration of agonist that produces 50% inhibition of forskolin-stimulated cAMP accumulation). The amount of cAMP in the sampleswas quantified against a cAMP standard curve. Forskolin (100 μM),stimulated cAMP formation in the absence of agonist was defined as100%. Results are reported as the mean±S.E.M. and statisticalsignificance was determined using one-way ANOVA at Pb0.05.

2.6. Eu-GTP-γS binding assays

Eu-GTP binding assay is a powerful high throughput alternative tothe well accepted 35S-GTPγS binding assay. The assay was performedin Acro-Well filter plates, essentially, as described previously(Engström et al., 2004) for membranes of CHO cells stably expressingthe human κ– and μ–opioid receptors. The reaction was started byaddingmembranes (4–9 μg protein/sample) to the assay solution. Theassay buffer (50 mM Tris–HCl, 1 mM EDTA, 1 mM dithiothreitol, 1 μMGDP for hMOR and 3 μM GDP for hKOR, 125 mM NaCl, and 5 mMMgCl2, pH 7.4) contained 0.1 nM to 200 nM of the test peptide andcontrol. A 30 min preincubation without label was followed by a30 min stimulation period after the addition of 10 nM Eu-GTP(Perkin-Elmer Life Sciences, Wallac, Turku, Finland; product code:AD0260). The reaction was terminated by vacuum filtration (MultiScreen Vacuum Manifold, Millipore) and the filter plate was washedfive times with 300 μl of ice-cold wash buffer (20 mM Tris–HCl,5mMMgCl2, 1 mMEDTA, pH 7.4) per well. The Eu-GTP retained on thefilter was then measured with a VICTOR2TM V Multilabel Counter(Perkin-Elmer Life Sciences, Wallac, Turku, Finland) at 340 nmexcitation/615 nm emission.

2.7. Data analysis

Cyclic AMP data were calculated as per the procedure given in thekit. The percent over basal binding was calculated according to theformula: the % Over Basal Binding = [{Stimulated Signal (Average)X100}/{Basal Signal (Average)}−100]. All the assays were per-formed in triplicate and data were analyzed by student t-test and one-way ANOVA in Origin version 7.1. A P valueb0.05 was consideredstatistically significant.

3. Results

3.1. Effects of acute YFa treatment on forskolin-stimulated cAMPformation in hKOR-CHO cells

YFa demonstrated forskolin-stimulated cAMP inhibition similar toDynA (1–13) in a concentration dependent manner (Fig. 2). At 10 nMconcentration, YFa significantly inhibited forskolin-stimulated cAMPformation; the inhibition was 23% (P=0.003); the peptide showedalmost 3-fold augmentation in inhibition on increasing the concen-tration to 20 nM. The inhibition of YFa reached saturation, 87–89% at60 nM (Fig. 2). DynA (1–13), an endogenous agonist of κ-opioidreceptors, demonstrated very high inhibition of forskolin-stimulated

cAMP accumulation at 10 nM concentration; it was 44% (P=0.003)and it reached the saturation, 89–91% at 50 nM. Both YFa andDynA (1–13) did not show any statistically significant change ininhibition when concentration was increased above 100 nM. Theseresults were further potentiated by IC50 values of YFa; log−7.826 nM(i.e. 14.9±1.477 nM; P=0.003) and DynA (1–13); log −7.990 nM(i.e. 11.9±1.231 nM; P=0.003) (Table 1). Furthermore, when YFawas incubated with κ-receptor specific antagonist, nor-Binaltorphi-mine (nor-BNI) in hKOR-CHO cells, it showed reversal of inhibitionactivity. It demonstrated residual inhibition approximately 33% evenat 100 nM; (P=0.003, Fig. 2).

3.2. Effects of acute YFa treatment on forskolin-stimulated cAMPformation in hMOR-CHO cells

YFa, at 10 nM concentration showed 17% (P=0.001) inhibition offorskolin-stimulated cAMP formation, which almost doubled whenconcentration was increased to 20 nM (Fig. 3). The peptide showedsaturation in inhibition, 69–71%, at 100 nM. However, morphine andDAMGO inhibited forskolin-stimulated cAMP formation more potent-ly, the inhibition being 37% and 43% at10 nM (P=0.001). Both theagonists demonstrated a more than 1.5-fold rise in inhibition whenconcentration was increased from 10 nM to 20 nM and showedsaturation in inhibition, 89–93% at 100 nM. The results were furtherconfirmed by IC50 values of YFa, DAMGO and morphine which werelog −7.413 nM (i.e. 38.6±7.442 nM), log −8.036 nM (i.e. 9.2±1.221 nM) and log −7.942 nM (i.e. 11.5±1.771 nM) ( P=0.001),

Fig. 3. % Inhibition of forskolin-stimulated cAMP formation in hMOR–CHO cells in acutetreatment of 30 m. Values represent mean±S.E.M. for three experiments performed intriplicate. IC50 values were calculated by one way ANOVA and data was significant withP=0.001. When YFa was incubated with mu specific antagonist Naloxonazine itdemonstrated the reversibility in activity (i.e. 25% at 150 nM, P=0.001).

Fig. 4. Effect of chronic treatment of morphine and DAMGO demonstrating superactivationof adenylyl cyclase in hMOR-CHO cells. Significant change in Cyclic AMP level was observedonly from 20 nM onwards. Both DAMGO and morphine showed maximum cAMPaccumulation i.e. 93% and 107% (P=0.03) at 150 nM, respectively. At the sameconcentration both the agonist demonstrated the maximum inhibition of cAMP accumu-lation between two successive concentrations within the treatment i.e. acute or chronic.

31K. Kumar et al. / European Journal of Pharmacology 650 (2011) 28–33

respectively (Table 1). Similar to hKOR cells, YFa showed reversibilityin inhibition activity when it was co-administered with μ–opioidreceptor specific antagonist, Naloxonazine. It showed residualinhibition approximately 25% even at 150 nM; (P=0.001, Fig. 3);however it did not match the level of κ-opioid receptors (Fig. 3).

3.3. Effect of Chronic YFa treatment on cAMP accumulation in hKOR andhMOR cells

Acute and chronic treatment of YFa did not show any significantchange in forskolin-stimulated cyclic AMP accumulation in hKOR andhMOR cells (Table 2). DynA (1–13) also demonstrated similar resultsin hKOR cells (Table 2).There was no loss of inhibition activity of YFaagainst cAMP accumulation in chronic treatment when comparedwith acute experiment in both the cell lines. In comparison to this,however, morphine and DAMGO demonstrated significant rise inforskolin-stimulated cAMP level in hMOR cells form 20 nM onwards(Fig. 4). It indicated an up-regulation of adenylyl cyclase. DAMGO andMorphine, in comparison to acute treatment, exhibited maximumcAMP accumulation at 150 nM in hMOR cells; it was 93% and 107%,(P=0.03, Fig. 4), respectively. However, cyclic AMP accumulationwas more in case of chronic morphine treatment in comparison toDAMGO.

Table 2Quantification of cAMP accumulation with acute and chronic treatment of YFa in hKORand hMOR cells and DynA (1–13) in hKOR cells.

Concentration nM YFa DynA(1–13)

hMOR-CHOa hKOR-CHOa hKOR-CHOa

30 min 20 h 30 min 20 h 30 min 20 h

10 710.7 707.7 604.7 604.7 339.6 338.620 674.3 677.3 316.5 315.5 243.8 244.850 511.9 513.9 208.0 210.0 174.0 172.0100 291.2 293.2 155.7 154.7 134.1 132.1150 273.1 275.1 102.2 104.2 93.8 95.8200 244.2 242.7 94.7 94.7 87.5 86.5250 234.8 231.1 86.1 83.1 76.2 75.2

a Values of cAMP accumulated at two time points i.e. acute and chronic are notsignificantly different and are the mean±S.E.M. of two sets of three experimentsperformed independently.

3.4. Eu-GTP-γS binding assay of hKOR-CHO membrane with YFa

In this study, Eu-GTP-γS binding assays of YFa and DynA (1–13)were conducted with hKOR-CHO membrane and receptor expressionwas assessed by western blotting (Fig. 1). Both the ligands wereincubated with KOR membrane as per the method and the respectiveconcentration response curves were obtained (Fig. 5). YFa showedvery pertinent interaction with hKOR membrane in terms of % overbasal binding similar to DynA (1–13). In a concentration response of6 nM to 10 nM, there was almost 1.5-fold increase in Eu-GTP-γS %over basal binding. DynA (1–13) showed a 1.5-fold increase in % overbasal binding (P=0.001) with a concentration enhancement of10 nM to 30 nM; however, YFa exhibited the increment of more than2-fold (P=0.003) and saturation point was exhibited at 60 nM. Therewas no significant divergence in Eu-GTP-γS % over basal bindingat 100 nM and above. EC50 values for YFa and DynA (1–13) werelog −7.942 nM (i.e. 11.5±1.576 nM, P=0.003) and log−8.096 nM(i.e. 8.0±1.331 nM, P=0.003), respectively (Table 3). When YFa andnor-BNIwere co-incubated with hKORmembrane, it demonstrated aninstant fall in Eu-GTP-γS % over basal binding. It showed a lowest

Fig. 5. Concentration dependent Eu-GTP-γS binding assay in YFa and DynA (1–13) withhKOR membrane in presence of 3 μM GDP, 125 mM NaCl, and 5 mM MgCl2. Valuesrepresent mean±S.E.M. for three experiments performed in triplicate. EC50 valueswere calculated by one way ANOVA and data was significant with P=0.001. When nor-BNI was pre incubated with hKOR membrane, YFa demonstrated an instant decline ofEu-GTP-γS %over basal binding which was maximum at 30 nM (P=0.001).

Table 3Eu-GTP-γS binding assay results showing EC50 values of YFa and control agonists forkappa and mu receptor in hKOR and hMOR CHO cells.

CHO-hKOR CHO-hMOR

Agonist EC50Log (nM)*[n=14]

Agonist EC50Log (nM)*[n=14]

DynA(1-13) −8.096 DAMGO −8.036YFa −7.942 Morphine −7.917YFa+nor-BNI Not significant YFa −7.701

DynA(1–13) Not significantYFa+Naloxonazine Not significant

*P values for hKOR and hMOR cells were statistically significant; (P=0.003) and(P=0.001). The values represent mean±S.E.M. for three experiments performed intriplicate. EC50 values were calculated by one-way ANOVA and data statisticallysignificant.

32 K. Kumar et al. / European Journal of Pharmacology 650 (2011) 28–33

residual binding of 7.94% even at 30 nM (P=0.00, Fig. 5), signifyingthe interaction of chimeric peptide with κ-opioid receptor.

3.5. Eu-GTP-γS binding assay of hMOR-CHO membrane with YFa

Similar to hKOR-CHO membrane assay, hMOR-CHO membraneassays were performed (Fig. 6). The receptor expression wasevaluated by western blotting (Fig. 1).

YFa showed almost 9-fold increase in binding against a concen-tration response of 6 nM to 10 nM, whereas the increase was almost3-fold (P=0.001) against a concentration increment of 10 nM to20 nM. DAMGO and morphine demonstrated Eu-GTP-γS bindingat the lowest experimental concentration of 1 nM and showed almost2-fold increase (P=0.001) in Eu-GTP-γS % over basal binding over arange of 6 nM to 20 nM. The EC50 values for YFa, morphine andDAMGO were log −7.701 nM (i.e. 19.9±2.447 nM, P=0.001),log −7.917 nM (i.e. 12.1±1.331 nM, P=0.001) and log −8.013 nM(i.e. 9.6±1.773 nM, P=0.001), respectively (Table 3). DAMGO andmorphine reached the saturation in Eu-GTP-γS % over binding at60 nM. Interestingly YFa showed the point of saturation in Eu-GTP-γS% over basal binding at 100 nM; it was 154–179%. YFa demonstrated arelatively lower binding affinity towards hMOR-CHO cells in terms ofEu-GTP-γS % over basal binding. When YFa and Naloxonazine were

Fig. 6. Concentration dependent Eu-GTP-γS binding assay in YFa DynA (1–13),Morphine and DAMGO with hMOR membrane in presence of 1 μM GDP, 125 mM NaCl,and 5 mM MgCl2. Values represent mean±S.E.M. for three experiments performed intriplicate. EC50 values were calculated by one way ANOVA and data was significant withP=0.003. When Naloxonazine was pre incubated with hMOR membrane, YFademonstrated a decline of Eu-GTP-γS %over basal binding which was maximum at30 nM (P=0.003).

co-incubated with hMOR membrane, it demonstrated an instantdecline in Eu-GTP-γS % over basal binding. It showed a lowest residualbinding of 13.71% even at 30 nM (P=0.001, Fig. 6), indicating theinteraction of chimeric peptide with μ–opioid receptor, also. Amongthe two binding assays, YFa demonstrated more reversibility in Eu-GTP-γS % over basal binding in hKOR cells.

4. Discussions

The present investigation was undertaken with an aim to assessthe stimulation of Eu-GTP-γS binding and inhibition of forskolin-stimulated cAMP formation by chimeric peptide YFa in cell linesexpressing μ– and κ–opioid receptors. In our previous reports onchimeric peptide YFa involving differential expression of μ–, δ– and κ–receptors we had demonstrated that κ-opioid receptor was involvedin YFa antinociception with no tolerance development during 6 daysof in vivo (i.p. administration in rats) treatment (Vats et al., 2008).Further in our cross tolerance studies (Gupta et al., 2008) on YFa, thetolerance and morphine induced cross tolerance were also notobserved to its analgesic effect even at day 5 after pretreatment (i.p.administration in mouse) with either YFa or morphine for 4 days.These findings prompted us to further investigate whether YFa, inacute and chronic treatment, affected the forskolin-stimulated cAMPformation and Eu-GTP-γS binding in cell line expressing hKOR andhMOR to strengthen our studies on YFa.

YFa exhibited concentration dependent inhibition of cAMPformation and was more selective to κ–receptor than μ–receptorand the inhibition was comparable to DynA (1–13). In proximity IC50values of YFa and DynA (1–13) also substantiated the affinity of thepeptide for κ–opioid receptor. Whereas, DAMGO andmorphine, in thesame experiment, demonstrated cAMP inhibition at lower concentra-tion and also revealed comparable IC50 values. To further authenticateour results, specific antagonist studies on forskolin-stimulated cAMPformation were performed and it was observed that the nor-BNI andNaloxonazine treatment of κ–and μ–receptors completely antagonizedthe YFa activity on forskolin-stimulated cAMP formation in hKOR-CHOand hMOR-CHO cells. Interestingly, the reversibility was more signif-icant in hKOR-CHO than in hMOR-CHO. This further strengthenedthat YFa hadmore inclination towards κ- receptor than μ– receptor. TheYFa's interaction with κ– and μ–receptors could be attributed to NPFFreceptor that modulates μ–receptor activity (Roumy et al., 2007). Theother possible explanation could be that YFa contained, besides opioidsequence, antiopioid sequence (RF-amide) at C-terminal which mightinteract with NPFF thereby modulating μ–receptor activity. To resolvethese issues on YFa, we are further continuing with our investigations.Nonetheless, the present findings are well in agreement with ourprevious studies on YFa on differential mRNA expression of opioidreceptors (Vats et al., 2008) and cross tolerance (Gupta et al., 2008).

Chronic treatment of μ–receptor expressing cells in cultureproduces a compensatory up-regulation of cAMP pathway and isreported as the reason for tolerance development (Koch et al., 2005;Avidor-Reiss et al., 1995; Zhanget al., 2009; Luttrell and Luttrell, 2004).To further validate the tolerance free analgesia of YFa, chronictreatment of YFa in hKOR and hMOR cells was examined, howeverforskolin-stimulated cAMP accumulation, in both hKOR and hMORcells, was comparable to acute treatment. Whereas, morphine andDAMGO demonstrated significant rise in forskolin-stimulated cAMPformation in hMOR cells. Thus, themajor findings of the present studyof insignificant alterations in cAMP accumulation in acute as well aschronic treatment in cells expressing hKOR and hMOR contrary toDAMGO and morphine clearly demonstrated an up regulation ofadenylyl cyclase which could be the basis of tolerance development incase of DAMGO andmorphine (Zhang et al., 2009; Luttrell and Luttrell,2004; Nestler and Aghajanian, 1997; Waldhoer et al., 2004; Xu et al.,2007; Roumy et al., 2007). Furthermore, the observed effect, accordingto the recent reports, may be attributed to the existence of opioid

33K. Kumar et al. / European Journal of Pharmacology 650 (2011) 28–33

receptors as homo/hetero dimer which may facilitate YFa in multipleopioid receptors binding (Snook et al., 2008; Franco et al., 2008).Moreover, the simultaneous targeting of ligand YFa to multiple opioidreceptors may produce analgesia without the development oftolerance (Dietis et al., 2009).

The Eu-GTP-γS binding assays further confirmed YFa affinitytowards κ–receptor compared to μ–receptor. It was evident by EC50

values of DAMGO, morphine and YFa and also by Eu-GTP-γS assayswith YFa in presence of the specific antagonists of hKOR and hMORnamely norBNI and Naloxonazine, respectively. Overall, YFa demon-strated more potent activity with κ–receptor when compared withμ–opioid receptor. Additionally, fall in Eu-GTP-γS bindings of YFanor-BNI and Naloxonazine, with hKOR and hMOR membrane, led tothe interaction of YFa with both κ– and μ–opioid receptors. Thegreater decline in Eu–GTP–γS binding of YFa in hKOR cells at allconcentrations further ascribed the more selectivity for κ–opioidreceptors. Thus, YFa, at all concentrations i.e. lowest to highestdemonstrated more potent affinity toward κ–receptors, however athigher concentration it showed binding with μ–receptor as well. TheEu–GTP–γS binding studies further validated the multiple receptorbinding of YFa and hence the rationale in favor of tolerance freeanalgesia (Dietis et al., 2009). These findings corroborate with ourprevious studies on YFa (Gupta et al., 1999, 2008; Vats et al., 2008).

5. Conclusion

To conclude, YFa interacted through κ–opioid receptor to inhibitforskolin-stimulated cyclic AMP formation and at a higher concen-tration with μ–receptor as well. In addition YFa, unlike morphine andDAMGO, did not exhibit cAMP super activation after chronictreatment. Eu–GTP–γS binding studies of YFa also proved it to be anagonist of κ–opioid receptor and also of μ–opioid receptor but athigher concentration. The tolerance free antinociception of YFa withκ– and μ–receptors may be accredited to multiple bindings.

Acknowledgements

Council of Scientific and Industrial Research India financiallysupported this study through NWP-013 project of NCL-IGIB. Theauthors are thankful to Prof. Richard B Rothman, NIH Maryland, USAfor his kind gift of cell lines stably expressing κ– and μ–opioidreceptor. We are also thankful to Dr. Naveen Khanna ICGEB Delhi forextending his support for this work.

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