ligand-specific regulation of the endogenous mu-opioid receptor

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 501086 9 pageshttpdxdoiorg1011552013501086

Research ArticleLigand-Specific Regulation of the EndogenousMu-Opioid Receptor by Chronic Treatment withMu-Opioid Peptide Agonists

Marianna Muraacutenyi1 Resat Cinar1 Orsolya Keacutekesi1 Erika Birkaacutes1 Gabriella Faacutebiaacuten1

Beaacuteta Bozoacute1 Andraacutes Zentai12 Geacuteza Toacuteth1 Emese Gabriella Kicsi1 Moacutenika Maacutecsai2

Roberta Dochnal2 Gyula Szaboacute2 and Maacuteria Szuumlcs1

1 Institute of Biochemistry Biological Research Center Hungarian Academy of Sciences PO Box 521 Szeged 6701 Hungary2Department of Pathophysiology Faculty of Medicine University of Szeged Szeged Hungary

Correspondence should be addressed to Maria Szucs szucsmhotmailcom

Received 30 April 2013 Revised 22 August 2013 Accepted 6 September 2013

Academic Editor Eiichi Kumamoto

Copyright copy 2013 Marianna Muranyi et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Since the discovery of the endomorphins (EM) the postulated endogenous peptide agonists of the mu-opioid receptors severalanalogues have been synthesized to improve their binding and pharmacological profiles We have shown previously that anew analogue cis-1S2R-aminocyclohexanecarboxylic acid2-endomorphin-2 (ACHC-EM2) had elevated mu-receptor affinityselectivity and proteolytic stability over the parent compound In the present work we have studied its antinociceptive effectsand receptor regulatory processes ACHC-EM2 displayed a somewhat higher (60) acute antinociceptive response than the parentpeptide EM2 (45) which peaked at 10min after intracerebroventricular (icv) administration in the rat tail-flick test Analgesictolerance developed to the antinociceptive effect of ACHC-EM2 upon its repeated icv injection that was complete by a 10-daytreatment This was accompanied by attenuated coupling of mu-sites to G-proteins in subcellular fractions of rat brain Also thedensity of mu-receptors was upregulated by about 40 in the light membrane fraction with no detectable changes in surfacebinding Distinct receptor regulatory processes were noted in subcellular fractions of rat brains made tolerant by the prototypic fullmu-agonist peptide DAMGO and its chloromethyl ketone derivative DAMCKThese results are discussed in light of the recentlydiscovered phenomenon that is the ldquoso-called biased agonismrdquo or ldquofunctional selectivityrdquo

1 Introduction

Recently discovered endomorphin-1 (Tyr-Pro-Trp-Phe-NH2

EM1) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2 EM2)

which display high affinity and selectivity to mu-opioidreceptors proved to be effective against neuropathic andinflammatory pain with reduced side effects [1 2] Besidestheir essential pharmacophoric groups (Tyr1 Phe3 and theamidated C-terminus) they have a proline in the secondposition that serves as a stereochemical spacer Substitutionof Pro2 by alicyclic beta-amino acids has been shown to causeprofound changes in bioactive conformation proteolytic sta-bility and pharmacological activity [3] Endomorphin-2 con-taining cis-(1R2S)-ACPC2 residue (ACHC-EM2) displayed

higher binding affinity improved mu-receptor selectivityand enhanced proteolytic stability compared to the parentpeptide [3 4] These features make it a promising tool tostudy the conformational requirements of peptide binding tomu-receptor

Opioids are among the most commonly used analgesicsbut their clinical use is limited by the development of variousunwanted side effects such as tolerance physical dependenceand respiratory suppression It has become apparent thatanalgesic tolerance and dependence are very complex phe-nomena which involve changes at the cellular neuronal andsystem levels [5ndash9] Traditionally receptor desensitization(uncoupling the receptor and G-protein) internalization(trafficking the cell surface receptors into an intracellular

2 BioMed Research International

compartment) andor downregulation (decrease in the totalnumber of receptors) have been proposed as possible mech-anisms for tolerance [6ndash15] Importantly while endogenousopioid peptides efficiently desensitized and internalized mu-opioid receptors the highly addictive morphine failed toinducemeasurable changes [11ndash18]There are conflicting datawhether there is a linear relationship between the intrinsicefficacy of a ligand and its ability to promote receptor interna-lization [19ndash22] Whistler and colleagues have suggested thatreceptor endocytosis plays a protective role in reducing thedevelopment of tolerance [8 15 23] Another new hypothesisis that opioid tolerance may also result from amplification orinduction of an opioid receptor-coupled signal transductionpathway that is either poorly expressed or absent from opioidnaive tissue [24] Very recently it has been revealed thatthe mu-opioid receptors besides other G-protein coupledreceptors display ldquofunctional agonismrdquo also called ldquoagonist-directed traffickingrdquo or ldquobiased agonismrdquo implying that differ-ent agonists while acting at the same receptor site can inducedistinct molecular changes and activate distinct downstreamresponses [25 26]

Most works on opioid receptor trafficking were carriedout in various in vitro cell models The limitations of thesemodels are obvious including differences in cellular milieuand receptor expression levels The study of endogenousopioid receptors using in vivo models has produced someinteresting results that could not have been anticipated invitro [27] As part of a comprehensive work using variousmu-opioid ligands of distinct efficacy chemical nature andabuse potential we attempt to correlate receptor regulatorychanges with analgesic tolerance It has been shown thatsustained morphine treatment of rats leading to analgesictolerance did not change the density or G-protein couplingof the surface mu-opioid receptors However significantintracellular changes including upregulation of the mu-sites and their cognate G-proteins were noted in the lightmembrane fraction [28] In addition we have described that14-methoxymetopon an extremely potent centrally actingmu-opioid specific analgesic with low tolerance physicaldependence and other side effects when given to rats eitheracutely or chronically did not change the binding or G-protein signaling of mu-opioid receptors in rat brain subcel-lular membranes [29] We have hypothesized that whereassurface opioid receptors and their cognate G-proteins medi-ate the acute effect of opioids intracellular events may play acrucial role in the long-term changes elicited by chronic drugexposure [28]

In the present work we have examined the new endo-morphin analogue ACHC-EM2 for its antinociceptiveeffect and receptor regulatory changes after acute andprolonged in vivo treatments The model system usedis based on subcellular fractionation of rat brains fol-lowed by ligand binding and functional measurementsDetailed characterization of the subcellular fractions bymarker enzymes electron microscopy and receptor bind-ing experiments has been reported [28ndash31] To assesschanges in the intracellular distribution of mu-opioid recep-tors subcellular fractionation was combined with radioli-gand binding measurements The degree of desensitization

was estimated by ligand-stimulated guanosine-51015840-O-(3-[120574[35S]hio)triphosphate [35S]GTP120574S functional assays It isconcluded that the regulatory changes accompanyingACHC-EM2 analgesic tolerance are distinct from those of theprototypic mu-agonist peptide DAMGO (Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol) and its chloromethyl derivative DAMCK(Tyr-D-Ala-Gly-(NMe)Phe-CH

2Cl)

2 Materials and Methods

21 Chemicals Endomorphin derivatives were synthesizedas published [3] DAMGO and [3H]DAMGO (36 Cimmol)were prepared and kindly provided to us by Drs Farkasand Toth (Biological Research Center Szeged Hungary)or purchased from Multiple Peptide System (San DiegoCA USA) DAMCK was synthesized as published [32] byAnna Magyar (ELTE Budapest Hungary) [35S]GTP120574S wasobtained from Isotope Institute Ltd (Budapest Hungary)Tris(hydroxymethyl)aminomethane (Tris free base)sodium chloride (NaCl) ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA) guanosine 51015840-diphosphate sodiumsalt (GDP) guanosine 51015840-triphosphate sodium salt (GTP)guanosine 51015840-[120574-thio]triphosphate tetralithium salt (GTP-120574-S-Li4) magnesium chloride hexahydrate (MgCl

2times 6H2O)

dithiothreitol sucrose and Nembutal were purchased fromSigma-Aldrich (St Louis MO USA) Bradford reagent wasfrom Bio-Rad Laboratories (Hercules CA USA)

22 Animals Wistar rats (250ndash500 g) were used They werekept under a standard light-dark cycle with food and wateravailable ad libitum The animals were kept and treatedaccording to the rules of the Ethical Committee for theProtection of Animals in Research (University of Szeged andBiological ResearchCenter of theHungarianAcademy of Sci-ences Szeged Hungary) All efforts were made to minimizetheir suffering during treatmentsThe animalswere randomlyassigned to treatment groups (119899 = 4dosetreatment)

23 Surgery For intracerebroventricular (icv) peptide admin-istration the rats were implanted with a stainless steelLuer cannula (10mm long) aimed at the right lateral cere-bral ventricle under Nembutal (35mgkg intraperitoneally)anesthesia [32] The stereotaxic coordinates were 02mmposterior 17mm lateral to the bregma and 37mm deepfrom the dural surface according to the atlas of Pellegrinoet al [33] Cannulas were secured to the skull with dentalcement and acrylate The experiments were started 5 daysafter icv cannulation All icv treatments were delivered tofreely moving animals to prevent unspecific effects Uponconclusion of the experiments 10 120583L of methylene blue wasinjected into the ventricle of decapitated animals and theposition of the cannula was inspected visually Animals withimproper cannula placement were excluded from the finalstatistical analysis

24 In Vivo Opioid Treatments The peptides were dissolvedin artificial cerebrospinal fluid (CSF) and injected in a volumeof 2 120583L Endomorphins were administered either acutely at

BioMed Research International 3

the indicated doses (100 pgndash20120583g) or chronically at 20120583g for10 days (twice daily) and tested at the indicated times Vehiclecontrol groups obtaining CSF were used in all experimentsand no changes were detected in the antinociceptive responseof the control group DAMGO and DAMCK were injectedat 10 120583g twice daily for 8 days Animals were killed 16 hrsafter drug treatments by decapitation immediately followedby subcellular fractionation

25 Tail-Flick (TF) Antinociceptive Assay The originalmethod of DrsquoAmour was used to determine analgesia in therat by measuring the time required to respond to a radiatingheat stimulus [32] A beam light was focused on the tip of thetail and the latency required for the rat to remove its tail wasdetermined before (baseline) and after drug administrationThe animals were tested at the times shown after injectionof the peptides The antinociceptive effect was expressedaccording to the equation (TF

119899minus TF119900) times 100TFmax minus TF119900

where TF119900is the tail-flick latency before drug administration

TF119899is the value of a repeated corresponding measurement

after drug injection and TFmax indicates the cutoff timewhich was set at 10 sec in our assays Statistical analysis of thedata of tail-flick antinociceptive test was made by ANOVAFor significant ANOVA values groups were compared byTukeyrsquos test for multiple comparisons with unequal cell sizeA probability level 119875 lt 005 was accepted to label significantdifferences

26 Crude Rat Brain Membranes Crude brain membraneswere prepared as published [34] Briefly pooled brain tissueswithout cerebella of four rats were washed with ice-coldbuffer and their weight was measured They were homoge-nized in 30 volumes (vw) of ice-cold 50mMTris-HCl buffer(pH 74) Homogenates were centrifuged at 20000timesg for25min and the resulting pellets suspended in buffer andspun again Pellets were taken up in the original volumeof buffer and incubated for 30min at 37∘C followed bycentrifugation at 20000timesg for 25min The supernatantswere carefully discarded and the final pellets were takenup in 5 volumes (vw) of 50mM Tris-HCl buffer (pH 74)containing 032M sucrose Appropriate membrane aliquotswere stored at minus80∘C for several weeks Prior to the bindingexperiments an appropriate aliquot was thawed dilutedwith 5-fold Tris-HCl buffer and centrifuged at 20000timesg for25min to remove sucrose The resulting pellets were takenup in 50mM Tris-HCl buffer (pH 74) to yield in 03ndash05mgmembrane proteinmL

27 Subcellular Fractionation of Rat Brains Subcellular frac-tions of rat brains were purified as published [28 29] Brieflyfresh forebrains of four rats were pooled gently homogenizedin 10 volumes (vw) of ice-cold buffer (5mM Tris-HCl pH74 50 120583M CaCl

2 05mM dithiothreitol) and supplemented

with 10 sucrose All sucrose solutions were made in theabove buffer The homogenate was spun at 1000timesg for10min The resulting pellet was resuspended in the abovebuffer and spun again The combined supernatants werespun at 12000timesg for 20min The pellets were suspended in

10 sucrose and subjected to consecutive centrifugations at20000timesg for 25min and 14000timesg for 20min twice resultingin crude synaptic plasma membranes (SPM) The resultingpellets were lysed followed by fractionation on a 10 285and 34 sucrose density step gradient that was spun at100000timesg for 2 h Highly enriched SPMs were obtainedfrom the 2834 interface Crude microsomal (MI) fractionswere obtained from the 12000timesg supernatant by consecu-tive centrifugations at 20000timesg for 25min and 100000timesgfor 1 h MI fractions were purified on a 10 and 285sucrose step gradient centrifuged at 100000timesg for 2 h andcollected at 10285 interface SPM and MI fractions werediluted threefold with 50mM Tris-HCl buffer and pelletedat 100000timesg for 1 h to remove sucrose The resulting pelletswere taken up in 50mM Tris-HCl (pH 74) buffer to yield in03ndash08mg membrane proteinml and were freshly used

28 Protein Concentration The protein content of the mem-brane preparations was determined by the method of Brad-ford using bovine serum albumin as a standard [35]

29 [3H]DAMGO Binding Assay Briefly homologousdisplacement assays were performed by incubating[3H]DAMGO (asymp1 nM) with 11 concentrations of unlabeledDAMGO (10minus10ndash10minus5M) and the membrane suspension(200ndash300120583g protein) in 50mM Tris-HCl (pH 74) bufferin a final volume of 1mL as described [28 29] The tubeswere incubated at 25∘C for 1 h The reaction was stopped byvacuum filtration through Whatman GFC glass fiber filters(Whatman Maidstone England) using a Brandel M24-RCell Harvester (Brandel Gaithersburg MD USA) Filterswere rapidly washed with 3 times 5mL ice-cold 50mM Tris-HCl(pH 74) buffer air-dried and counted in a toluene-basedscintillation cocktail in a Wallac 1409 scintillation counter(Wallac Turku Finland) All experiments were performedin duplicate and repeated at least three times Curves wereconstructed and analyzed by means of the GraphPad Prism4 program (GraphPad Software Inc San Diego CA USA)to obtain 119870

119863(dissociation constant) and 119861max (receptor

density) values The data reported are means plusmn SEM

210 Ligand-Stimulated [35S]GTP120574S Functional Assay Theassay was performed as published [28 29] with slight mod-ifications Briefly rat brain membrane fractions (asymp10 120583g ofprotein) were incubated with [35S]GTP120574S (005 nM) and 5-6 concentrations (10minus9minus10minus4M) of opioid peptides in thepresence of 100120583M GDP in Tris-EGTA (50mM Tris-HCl1mM EGTA and 5mM MgCl

2 pH 74) buffer in a total

volume of 1mL for 60min at 30∘C Nonspecific binding wasdetermined with 10 120583M GTP120574S and subtracted Bound andfree [35S]GTP120574S were separated by vacuumfiltration throughWhatman GFF filters using a Brandel M24-R Cell Harvester(Brandel Gaithersburg MD USA) Filters were rapidlywashed with 3 times 5mL ice-cold 50mM Tris-HCl (pH 74)buffer air-dried and counted in a toluene-based scintillationcocktail in aWallac 1409 scintillation counter (Wallac TurkuFinland) All assays were performed in triplicate and repeated


0

10

20

30

40

50

60

10 15 20 40(min)

Ant

inoc

icep

tive e

ffect

()

Control100 pg10 ng

1120583g20120583g

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

(a)

10 15 20 400

10

20

30

40

50

60

Ant

inoc

icep

tive e

ffect

()

(min)

Control100 pg10 ng

1120583g20120583g

lowast

lowast

lowast

lowast

lowast

lowast

lowast

(b)

Figure 1 Time-course of the acute antinociceptive effect of graded doses of EM2 (a) and ACHC-EM2 (b) in rat tail-flick assay The peptideswere administered icv and assayed as described in Methods Control groups received CSF Data are shown as mean plusmn SEM 119899 ge 6 Statisticalsignificance was determined by ANOVA and set at lowast119875 lt 005 compared to appropriate control values

0

10

20

30

40

50

1 3 5 7 9Days

Ant

inoc

icep

tive e

ffect

()

lowast

lowast

lowast

(a)

0

10

20

30

40

50

1 3 5 7 9Days

Ant

inoc

icep

tive e

ffect

()

lowast

lowast

lowast lowast

(b)

Figure 2Development of antinociceptive tolerance to chronicACHC-EM2given at 20120583g2 120583L twice daily for the indicated time periods Ratswere tested in the tail-flick assay 5min (a white columns) and 15min (b striped columns) after injection Mean plusmn SEM 119899 = 7 Significancewas determined by ANOVA lowast labels 119875 lt 005 compared to the first day

at least three times Curves were constructed and analyzed byfitting sigmoidal dose-response curves using the GraphPadPrism 4 program (GraphPad Prism Software Inc) to obtainpotency (EC

50 concentration of the ligand to give half-

maximal effect) and efficacy (119864max maximal stimulationover basal activity) values Basal activities were measured inthe absence of opioid ligands and set as 0The data reportedare means plusmn SEM

3 Results

31 Antinociceptive Effect of ACHC-EM2 after Acute Treat-ments In the first part of the study the antinociceptive effectof the new ligand ACHC-EM2 was evaluated and comparedto that of its parent peptide EM2 in tail-flick tests Ratswere injected icvwith different doses of the peptides between100 pg and 20120583g as indicated in Figure 1 Animals treatedwith lower doses of ACHC-EM2 displayed a concentration-dependent increase (20ndash50) in analgesic response com-pared to base-line values The acute antinociceptive effect

peaked around 10ndash15min followed by a rapid decreasewhich dissipated by 40min following injection (Figure 1)Higher doses (1 and 20 120583g) of ACHC-EM2 displayed around60 antinociceptive responses which were somewhat higherthan those of the parent peptide EM2 (45) which peakedat an earlier time (10min)

32 Analgesic Tolerance Develops to the Antinociceptive Effectof ACHC-EM2 after Chronic Treatments In the next setof experiments rats were repeatedly injected twice dailywith ACHC-EM2 (20120583g icv) and the antinociceptive effectwas measured 5 and 15min following injection on varioustreatment days It can be seen that the antinociceptive effectgradually decreased with time compared to day 1 whichbecame statistically significant by day 5 and ceased by day 10showing the development of analgesic tolerance (Figure 2)

33 Potency and Efficacy of ACHC-EM2 in Activating G-Proteins In Vitro ACHC-EM2 was measured in the ligand-stimulated [35S]GTP120574S functional assay in crude rat brain


Table 1 Changes in DAMGO-stimulated [35S]GTP120574S binding induced by in vivo chronic opioid peptide treatments in rat brain subcellularfractions

Treatment119864max ( over basal) EC50 (nM)

SPM MI SPM MIControl Treated Control Treated Control Treated Control Treated

ACHC-EM2 95 plusmn 5 106 plusmn 5 85 plusmn 9 109 plusmn 2 44 plusmn 6 80 plusmn 8lowast

100 plusmn 18 179 plusmn 25lowast

DAMGO 112 plusmn 12 130 plusmn 14 60 plusmn 3 72 plusmn 9 87 plusmn 9 107 plusmn 14 101 plusmn 14 86 plusmn 2

DAMCK 112 plusmn 12 100 plusmn 9 60 plusmn 3 61 plusmn 17 87 plusmn 9 126 plusmn 22 101 plusmn 14 200 plusmn 42lowast

ACHC-EM2 DAMGO and DAMCK were chronically administered to rats as described in Methods Control animals received CSF Subcellular fractionationof brain homogenates to obtain synaptic plasma membrane (SPM) and microsomal (MI) fractions was performed Full concentration curves of DAMGOconsisting of 5-6 concentrations between 10minus8ndash10minus4M were measured in [35S]GTP120574S binding assay The parameters shown were obtained from nonlinearregression analysis using Graph Pad Prism 4 considering a sigmoidal dose response curve for DAMGO Results shown are as stimulation of [35S]GTP120574Sbinding over basal values (ie binding in the absence of DAMGO) Data are mean plusmn SEM of 3ndash6 independent experiments each performed in triplicateSignificant difference between the appropriate values in control and treated membrane fractions was determined by the Studentrsquos 119905-test and set as lowast119875 lt 005

0

20

40

60

80

100

120

DAMGOEM-2ACHC-EM2

log[Ligand] (M)

Stim

ulat

ion

(ove

r bas

al) (

)

minus11 minus10 minus9 minus8 minus7 minus6 minus5 minus4

Figure 3 Stimulation of [35S]GTP120574S binding by opioid peptidesin crude rat brain membranes Membrane homogenates (asymp 10 120583gof protein) were incubated with increasing concentrations (10minus9ndash10minus4M) of the indicated ligands and [35S]GTP120574S (005 nM) asdescribed in Methods except that 30 120583MGDP was used The curveswere fit and drawn by Graph Pad Prism 4 computer programResults are shown as stimulation of [35S]GTP120574S binding overbasal values (ie binding in the absence of opioid peptides) Eachvalue represents the mean plusmn SEM of at least three independentexperiments performed in triplicate Nonvisible SEM is within thesymbol

membranes and compared to that of EM2 and the prototypicfull agonist DAMGO Full dose-response curves of the threepeptides were assessed between 10minus9minus10minus4Mand efficacy andpotency values were determined (Figure 3) The 119864max valueof DAMGO was set as 100 by definition The efficacy wassignificantly lower for both ACHC-EM2 and EM2 with 119864maxvalues of 717 plusmn 11 and 525 plusmn 08 respectively Thesedata show that both ACHC-EM2 and EM2 behave as partialagonists in this assay The potency of the three peptidesin stimulating G-protein activation was very similar withlog EC

50value of ACHC-EM2 being 637 plusmn 003

34 Changes in G-protein Stimulation due to Chronic Treat-ments with Mu-Opioid Peptide Agonists Brains exposed torepeated agonists treatments leading to analgesic tolerance orvehicle were simultaneously processed in every experimentControl and treated brain homogenates were subjected tosubcellular fractionation to obtain highly purified SPM andMI membrane fractions Functional coupling of mu-opioidreceptors to G-proteins was examined by measuring theability of DAMGO added to the membrane fractions in vitroto facilitate [35S]GTP120574S binding (Table 1) The potencies ofDAMGO were 44 plusmn 6 nM and 100 plusmn 18 nM and efficaciesof 95 plusmn 5 and 85 plusmn 9 in control (vehicle administered)SPM and MI respectively Chronic treatment with ACHC-EM2 resulted in a significant shift of the dose-response curveof DAMGO to the right accordingly the EC

50values of

DAMGO increased to 80 plusmn 8 nM and 179 plusmn 25 nM in treatedSPM and MI respectively Chronic treatment with DAMCKalso resulted in attenuated G-protein coupling in the MIfraction There was a similar tendency in the SPM whichhowever did not reach a statistically significant level ChronicDAMGO treatment did not cause any change in [35S]GTP120574SincorporationThe efficacies reflected in the 119864max values didnot change due to any treatments (Table 1)

35 Changes of Receptor Densities due to Chronic Treat-ments with Mu-Opioid Peptide Agonists Changes in thebinding parameters of mu-opioid receptors were measuredin [3H]DAMGO homologous displacement experiments(Table 2) As expected the 119870

119863values were not significantly

changed due to acute or chronic ACHC-EM2 treatmentsAcute injection of a single dose (20120583g) of the endomorphinderivative did not change the 119861max values in any fractionsNotably chronic treatment with this peptide resulted in asignificant (42) increase in the density of mu-sites in theMI fraction (Table 2 Figure 4(a)) On the contrary chronictreatments with the full agonist DAMGO resulted in a 22decrease in the density of surface mu-sites (Figure 4(b))Similar effect was detected with the chloromethyl ketonederivative of DAMGO DAMCK with a concomitant 40increase in the MI fractions (Figure 4(c))


0

10

20

30

40

50

60lowast

[3H

] DA

MG

O b

indi

ng (

chan

ge)

(a)

0

20

40

60

minus40

minus20

lowast[3H

] DA

MG

O b

indi

ng (

chan

ge)

(b)

0

20

40

60

minus40

minus20

lowast

[3H

] DA

MG

O b

indi

ng (

chan

ge)

lowast

(c)

Figure 4 Changes in receptor density (119861max) following chronic exposure of ACHC-EM2 (a) DAMGO (b) and DAMCK (c) SPM (whitecolumns) and MI (striped columns) fractions were prepared from whole brain The membrane suspensions (03mg protein) were incubatedwith 1 nM [3H]DAMGO for 60min at 25∘C in the absence (total binding) or in the presence of 10minus10ndash10minus5M of unlabeled DAMGO Resultsare expressed as change of protein in each fraction Mean plusmn SEM n = 3ndash6 significance was determined by 119905-test lowast119875 lt 005 compared tocontrol

Table 2 Changes in [3H]DAMGO binding induced by ACHC-EM2 treatments in rat brain subcellular fractions

Treatment119870119863(nM) 119861max (fmolmg protein)


ACHC-EM2 acute 50 plusmn 19 30 plusmn 11 23 plusmn 04 36 plusmn 09 298 plusmn 72 258 plusmn 45 410 plusmn 59 311 plusmn 74

ACHC-EM2 chronic 29 plusmn 12 13 plusmn 02 45 plusmn 24 25 plusmn 02 291 plusmn 82 286 plusmn 50 333 plusmn 78 462 plusmn 54lowast

ACHC-EM2was injected either acutely or chronically as described inMethods Control animals received CSF Subcellular fractionations of brain homogenatesto obtain synaptic plasma membrane (SPM) and microsomal (MI) membranes followed by [3H]DAMGO binding was performed as outlined in MethodsData represent the mean plusmn SEM of at least three independent experiments performed in duplicate Statistically significant differences due to either treatmentscompared to appropriate control values in each fraction were determined by Student 119905-test and set at lowast119875 lt 005

4 Discussion

In one part of the work we have described the pharmaco-logical features of the new endomorphin derivative ACHC-EM2 upon its icv administration in the rat tail-flick testAcutely it is slightly more potent than the parent peptideEM2 having 60 and 45 maximal antinociceptive effectvalues respectively (Figure 1) These results are consistentwith several investigations in acute pain tests showing thatendomorphins exhibited steady plateau at 40 of maximalantinociceptive effect [2 36ndash38] We have shown previouslythat the prototypic mu-agonist peptide DAMGO and itschloromethyl ketone derivative DAMCK display profoundantinociception of about 80 with distinct time-course Theeffect of DAMGOwas longer-lasting which peaked by about30min and followed by a rapid dissipation whereas DAMCKshowed an apparently irreversible antinociception [32] Thisis in accordance with its irreversible binding to mu-sites in invitro binding assays It is to be noted that we have intracere-broventricularly administrated EM2 and ACHC-EM2 whilethe antinociceptive effects were examined by using thetail-flick test mostly a spinal reflex Opioids are involvedin both ascending and descending components of painmodulation Given supraspinally multiple descending paincontrol pathways are involved in antinociception induced byopioid agonists Interestingly distinctmechanismsmediatingdescending pain controls for antinociception induced by

supraspinally administered EM1 and EM2 were described inthe mouse [39]

It has been revealed that full analgesic tolerance developsto the antinociceptive effect of 20120583g ACHC-EM2 by a 10-day treatment (icv twice daily) (Figure 2) For a comparisontolerance development was also tested for DAMGO andDAMCK Itwas found that behavioral tolerancewas completeby an 8-day icv injection of 10 120583g of the latter two peptides(twice daily (data not shown)) For further biochemicalstudies the doses of the peptides were designed to maximizetolerance and were not equieffective as performed with otherligands [19]

Since the ligand-stimulated [35S]GTP120574S functional assaymeasures receptor mediated G-protein activation it may bea viable tool to detect adaptations associated with toleranceat the mu-opioid receptor We have revealed that both EM2and its new derivative are partial agonists contrary to thefull agonist DAMGO in the ligand-stimulated [35S]GTP120574Sfunctional test in crude rat brain membranes (Figure 3) Thisis in an excellent agreement with our data in the tail-flickanalgesia test and recent work which showed that EM2 hasa much lower operational efficacy for G-protein mediatedresponses than DAMGO at native mu-opioid receptors inmature neurons [40] However tolerance to [35S]GTP120574Sincorporation following chronic opioid peptide treatmentswas not detected in all cases even when full analgesic


tolerance was manifested It was found that chronic ACHC-EM2 treatment decreased the EC

50of DAMGO by about

2-fold showing attenuated coupling of mu-sites to theircognate G-proteins in both SPM andMImembranes Similarchanges were observed upon prolonged DAMCK treatmentOn the contrary chronic DAMGO injection resulted in nosignificant changes in either the potency or in the efficacy ofG-protein signaling (Table 1) The question arises whether adifferent picture would have been obtained on ACHC-EM2-stimulated [35S]GTP120574S signaling Differences in the patternof G-protein activation have been reported for agonists atmu-opioid receptors including EM-1 versus EM2 and EM-2 versus DAMGO and morphine [41ndash43] In the presentwork we did not attempt to identify the G-protein subtypesinvolved in the effect of each ligand For detecting desen-sitization G-protein activation by the full agonist DAMGOmay be a better choice than the partial agonist ACHC-EM2whichmay induce either incomplete stimulation of the entiremu-opioid receptor population or full stimulation of only aportion of the entire receptor population Another reason fortesting DAMGO-stimulated [35S]GTP120574S binding is that thepresent study is part of a comprehensive work using variousmu-opioid ligands of distinct efficacy chemical nature andabuse potential to assess regulatory changes due to analgesictolerance and this set-up was used in our previous studies[28 29]

Ligand-specific changes were also detected for receptortrafficking (Figure 4) While chronic DAMGO and DAMCKdecreased the density of surface mu-receptors such changeswere not evident by ACHC-EM2 Instead the density of mu-sites increased by about 40 in the light membrane fractionof ACHC-EM2 tolerant brain homogenates compared to thatof vehicle-treatedMI fraction (Figure 4(c) Table 2) Previousworks from other laboratories have revealed region-specificreceptor adaptation in the brain [42 43] It is to be consideredwhether compounds injected via the icv route are able toreach the entire population of mu-opioid receptors in thecentral nervous system There are reports suggesting thatthe compounds may remain in the vicinity of the ventriclesOn the contrary icv administration of the opioid antagonistbeta-funaltrexamine was shown to reduce the density of mu-opioid receptors as measured by in situ autoradiography by40ndash50 throughout the brain with little regional variations[44] Since it is not possible to gain sufficient amount ofmembrane proteins in subcellular fractions of small tissueswe have measured mu-opioid receptor density in crudemembranes [3H]DAMGO binding was downregulated byabout 22 in the hippocampus upregulated by 33 in thespinal cord and did not significantly change in the cortex andbrainstem due to chronic ACHC-EM2 treatments (data notshown)

Overall distinct regulatory changes were noted for thethree opioid peptides albeit all caused full analgesic toler-ance This is in accordance with a growing number of datashowing that different opioid ligands can lead to varyingdegrees of receptor regulation The recently discovered phe-nomenon of ldquobiased agonismrdquo can provide the molecularbasis for the observed ligand-specific effects It is anticipated

that different agonists stabilize distinct active conformationsof the receptor thereby inducing distinct downstream signal-ing [25 26 45] A very recent paper has shown that endomor-phins seem to be biased toward arrestin recruitment over G-protein activation contrary to DAMGO [40] Morphine hasbeen shown to be biased toward 120573-arrestin2 regulation of themu-opioid receptors It was published that knocking-out thisprotein enhanced and prolonged morphine antinociceptionin the hot-plate test and attenuated tolerance [46] Althoughthe precise role of arrestins in the regulation of mu-opioidreceptors in neurons remains to be fully elucidated it mayexplain the observed ligand-specific effects in the presentwork and others Functional selectivity may open up newdirections for designing potent analgesics with less unwantedside effects

5 Conclusions

Chronic icv treatment with the partial agonist endomorphinanalog ACHC-EM2 resulted in the development of analgesictolerance in the rat tail-flick test This was accompanied bydesensitization of the mu-receptors in subcellular fractionsof rat brain Also the mu-receptors were upregulated byabout 40 in the light membrane fraction with no detectablechange in surface binding The prototypic full mu-agonistpeptide DAMGO and its chloromethyl ketone derivativeDAMCK also induced tolerance yet the accompanyingmolecular changes were different These results are in accor-dance with the recently discovered phenomenon that is theso-called ldquobiased agonismrdquo or ldquofunctional selectivityrdquo Thenew concept may help to identify new lead molecules whichcan substitute morphine in pain treatment with improvedpharmacological profile

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

This work was supported by NKTH DNT 082004 OTKAT 046434 ETT 355-08 and TAMOP 422-A-111KONV-2012-0052 Research GrantsThe technical assistance of IldikoNemeth Agnes Paal and Gusztav Kis is greatly acknowl-edged

References

[1] J E Zadina L Hackler L J Ge and A J Kastin ldquoA potent andselective endogenous agonist for the 120583-opiate receptorrdquo Naturevol 386 no 6624 pp 499ndash502 1997

[2] G Horvath M Szikszay C Tomboly and G BenedekldquoAntinociceptive effects of intrathecal endomorphin-1 and -2 inratsrdquo Life Sciences vol 65 no 24 pp 2635ndash2641 1999

[3] A Keresztes M Szucs A Borics et al ldquoNew endomorphinanalogues containing alicyclic beta-amino acids influence onbioactive conformation and pharmacological profilerdquo Journal ofMedicinal Chemistry vol 51 pp 4270ndash4279 2008


[4] C Tomboly A Peter and G Toth ldquoIn vitro quantitative studyof the degradation of endomorphinsrdquoPeptides vol 23 pp 1573ndash1580 2002

[5] E J Nestler B T Hope and K L Widnell ldquoDrug addiction amodel for the molecular basis of neural plasticityrdquo Neuron vol11 no 6 pp 995ndash1006 1993

[6] T Koch and V Hollt ldquoRole of receptor internalization in opioidtolerance and dependencerdquo Pharmacology and Therapeuticsvol 117 no 2 pp 199ndash206 2008

[7] J T Williams S L Ingram G Henderson et al ldquoRegulation of120583-opioid receptors desensitization phosphorylation internal-ization and tolerancerdquo Pharmacological Reviews vol 65 no 1pp 223ndash254 2013

[8] J L Whistler ldquoExamining the role of mu opioid receptorendocytosis in the beneficial and side-effects of prolongedopioid use from a symposium on new concepts in mu-opioidpharmacologyrdquoDrug andAlcohol Dependence vol 121 no 3 pp189ndash204 2012

[9] K Nagi and G Pineyro ldquoRegulation of opioid receptor sig-nalling implications for the development of analgesic toler-ancerdquoMolecular Brain vol 4 article 25 2011

[10] J R Arden V Segredo Z Wang J Lameh and W SadeeldquoPhosphorylation and agonist-specific intracellular traffickingof an epitope-tagged 120583-opioid receptor expressed in HEK 293cellsrdquo Journal of Neurochemistry vol 65 no 4 pp 1636ndash16451995

[11] N T Burford L M Tolbert and W Sadee ldquoSpecific G proteinactivation and 120583-opioid receptor internalization caused bymorphine DAMGO and endomorphin Irdquo European Journal ofPharmacology vol 342 no 1 pp 123ndash126 1998

[12] K Stafford A B Gomes J Shen and B C Yoburn ldquo120583-opioidreceptor downregulation contributes to opioid tolerance invivordquo Pharmacology Biochemistry and Behavior vol 69 no 1-2 pp 233ndash237 2001

[13] Y Qiu P Y Law and H H Loh ldquo120583-opioid receptor desensi-tization role of receptor phosphorylation internalization andresensitizationrdquo Journal of Biological Chemistry vol 278 no 38pp 36733ndash36739 2003

[14] C Sternini M Spann B Anton et al ldquoAgonist-selective endo-cytosis of 120583 opioid receptor by neurons in vivordquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 93 no 17 pp 9241ndash9246 1996

[15] L Martini and J L Whistler ldquoThe role of mu opioid receptordesensitization and endocytosis in morphine tolerance anddependencerdquoCurrent Opinion in Neurobiology vol 17 no 5 pp556ndash564 2007

[16] A Alt A Mansour H Akil F Medzihradsky J R Traynorand J H Woods ldquoStimulation of guanosine-51015840-O-(3-[35s]thio)triphosphate binding by endogenous opioidsacting at a cloned mu receptorrdquo Journal of Pharmacology andExperimental Therapeutics vol 286 no 1 pp 282ndash288 1998

[17] J L Whistler and M von Zastrow ldquoMorphine-activated opioidreceptors elude desensitization by 120573-arrestinrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol95 no 17 pp 9914ndash9919 1998

[18] K McConalogue E F Grady J Minnis et al ldquoActivation andinternalization of the 120583-opioid receptor by the newly discoveredendogenous agonists endomorphin-1 and endomorphin-2rdquoNeuroscience vol 90 no 3 pp 1051ndash1059 1999

[19] A Duttaroy and B C Yoburn ldquoThe effect of intrinsic efficacy onopioid tolerancerdquo Anesthesiology vol 82 no 5 pp 1226ndash12361995

[20] J McPherson G Rivero M Baptist et al ldquo120583-opioid receptorscorrelation of agonist efficacy for signalling with ability toactivate internalizationrdquo Molecular Pharmacology vol 78 no4 pp 756ndash766 2010

[21] M Pawar P Kumar S Sunkaraneni S Sirohi E AWalker andB C Yoburn ldquoOpioid agonist efficacy predicts the magnitudeof tolerance and the regulation of 120583-opioid receptors anddynamin-2rdquo European Journal of Pharmacology vol 563 no 1ndash3 pp 92ndash101 2007

[22] H Xu J S Partilla X Wang et al ldquoA comparison of nonin-ternalizing (Herkinorin) and internalizing (DAMGO) 120583-opioidagonists on cellular markers related to opioid tolerance anddependencerdquo Synapse vol 61 no 3 pp 166ndash175 2007

[23] A K Finn and J L Whistler ldquoEndocytosis of the mu opioidreceptor reduces tolerance and a cellular hallmark of opiatewithdrawalrdquo Neuron vol 32 no 5 pp 829ndash839 2001

[24] A R Gintzler and S Chakrabarti ldquoOpioid tolerance and theemergence of new opioid receptor-coupled signalingrdquo Molecu-lar Neurobiology vol 21 no 1-2 pp 21ndash33 2000

[25] T Kenakin ldquoFunctional selectivity through protean and biasedagonismwho steers the shiprdquoMolecular Pharmacology vol 72no 6 pp 1393ndash1401 2007

[26] J D Urban W P Clarke M von Zastrow et al ldquoFunctionalselectivity and classical concepts of quantitative pharmacologyrdquoJournal of Pharmacology and Experimental Therapeutics vol320 no 1 pp 1ndash13 2007

[27] Y Wang E J van Bockstaele and L Y Liu-Chen ldquoIn vivotrafficking of endogenous opioid receptorsrdquo Life Sciences vol83 no 21-22 pp 693ndash699 2008

[28] G Fabian B Bozo M Szikszay G Horvath C J Coscia andM Szucs ldquoChronic morphine-induced changes in mu-opioidreceptors and G proteins of different subcellular loci in ratbrainrdquo Journal of Pharmacology and ExperimentalTherapeuticsvol 302 pp 774ndash780 2002

[29] R Cinar O Kekesi E Birkas G Fabian H Schmidhammerand M Szucs ldquoLack of regulatory changes of mu-opioid recep-tors by 14-methoxymetopon treatment in rat brain Furtherevidence for functional selectivityrdquo Current PharmaceuticalDesign In press

[30] B L Roth M B Laskowski and C J Coscia ldquoEvidence fordistinct subcellular sites of opiate receptors demonstration ofopiate receptors in smooth microsomal fractions isolated fromrat brainrdquo Journal of Biological Chemistry vol 256 no 19 pp10117ndash10123 1981

[31] M Szucs and C J Coscia ldquoDifferential coupling of opioid bind-ing sites to guanosine triphosphate binding regulatory proteinsin subcellular fractions of rat brainrdquo Journal of NeuroscienceResearch vol 31 no 3 pp 565ndash572 1992

[32] G Szabo M Macsai E G Kicsi et al ldquoLong-lasting antinoci-ceptive effect ofDAMGOchloromethyl ketone in ratsrdquoPeptidesvol 20 no 11 pp 1321ndash1326 1999

[33] L J Pellegrino A S Pellegrino and A J Cushman StereotacticAtlas of the Rat Brain vol 8 Plenum New York NY USA 2ndedition 1979

[34] R Cinar T F Freund I Katona K Mackie and M SzucsldquoReciprocal inhibition of G-protein signaling is induced byCB1 cannabinoid and GABAB receptor interactions in rathippocampal membranesrdquo Neurochemistry International vol52 no 8 pp 1402ndash1409 2008

[35] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principle


of protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[36] B Przewłocka J Mika D Łabuz G Toth and R PrzewłockildquoSpinal analgesic action of endomorphins in acute inflam-matory and neuropathic pain in ratsrdquo European Journal ofPharmacology vol 367 no 2-3 pp 189ndash196 1999

[37] P Sanchez-Blazquez M Rodrıguez-Dıaz I DeAntonio andJ Garzon ldquoEndomorphin-1 and endomorphin-2 show differ-ences in their activation of mu opioid receptor-regulated Gproteins in supraspinal antinociception in micerdquo Journal ofPharmacology and Experimental Therapeutics vol 291 pp 12ndash18 1999

[38] H Xie J H Woods J R Traynor and M C Ko ldquoThe spinalantinociceptive effects of endomorphins in rats behavioral andGprotein functional studiesrdquoAnesthesia andAnalgesia vol 106no 6 pp 1873ndash1881 2008

[39] MOhsawaHMizoguchiMNaritaMChuHNagase andLF Tseng ldquoDifferential mechanisms mediating descending paincontrols for antinociception induced by supraspinally admin-istered endomorphin-1 and endomorphin-2 in the mouserdquoJournal of Pharmacology and Experimental Therapeutics vol294 no 3 pp 1106ndash1111 2000

[40] G Rivero J Llorente J McPherson et al ldquoEndomorphin-2 abiased agonist at the 120583-opioid receptorrdquo Molecular Pharmacol-ogy vol 82 no 2 pp 178ndash188 2012

[41] C S Breivogel D E Selley and S R Childers ldquoAcute andchronic effects of opioids on 120575 and 120583 receptor activation of Gproteins in NG108-15 and SK-N-SH cell membranesrdquo Journal ofNeurochemistry vol 68 no 4 pp 1462ndash1472 1997

[42] P Sanchez-Blazquez P Gomez-Serranillos and J GarzonldquoAgonists determine the pattern of G-protein activation inmu-opioid receptor-mediated supraspinal analgesiardquo BrainResearch Bulletin vol 54 no 2 pp 229ndash235 2001

[43] J Garzon M Castro and P Sanchez-Blazquez ldquoInfluence of Gzand Gi2 transducer proteins in the affinity of opioid agonists tomu receptorsrdquo European Journal of Neuroscience vol 10 no 8pp 2557ndash2564 1998

[44] T J Martin S I Dworkin and J E Smith ldquoEffects ofintracerebroventricular administration of 120573-funaltrexamine on[3H]DAMGObinding to rat brain sectionsrdquo Journal of Pharma-cology and Experimental Therapeutics vol 267 no 1 pp 506ndash514 1993

[45] E Reiter S Ahn A K Shukla and R J LefkowitzldquoMolecular mechanism of 120573-arrestin-biased agonism at seven-transmembrane receptorsrdquoAnnual Review of Pharmacology andToxicology vol 52 pp 179ndash197 2012

[46] L M Bohn R J Lefkowitz R R Gainetdinov K Peppel MG Caron and F T Lin ldquoEnhanced morphine analgesia in micelacking 120573-arrestin 2rdquo Science vol 286 no 5449 pp 2495ndash24981999

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


compartment) andor downregulation (decrease in the totalnumber of receptors) have been proposed as possible mech-anisms for tolerance [6ndash15] Importantly while endogenousopioid peptides efficiently desensitized and internalized mu-opioid receptors the highly addictive morphine failed toinducemeasurable changes [11ndash18]There are conflicting datawhether there is a linear relationship between the intrinsicefficacy of a ligand and its ability to promote receptor interna-lization [19ndash22] Whistler and colleagues have suggested thatreceptor endocytosis plays a protective role in reducing thedevelopment of tolerance [8 15 23] Another new hypothesisis that opioid tolerance may also result from amplification orinduction of an opioid receptor-coupled signal transductionpathway that is either poorly expressed or absent from opioidnaive tissue [24] Very recently it has been revealed thatthe mu-opioid receptors besides other G-protein coupledreceptors display ldquofunctional agonismrdquo also called ldquoagonist-directed traffickingrdquo or ldquobiased agonismrdquo implying that differ-ent agonists while acting at the same receptor site can inducedistinct molecular changes and activate distinct downstreamresponses [25 26]

Most works on opioid receptor trafficking were carriedout in various in vitro cell models The limitations of thesemodels are obvious including differences in cellular milieuand receptor expression levels The study of endogenousopioid receptors using in vivo models has produced someinteresting results that could not have been anticipated invitro [27] As part of a comprehensive work using variousmu-opioid ligands of distinct efficacy chemical nature andabuse potential we attempt to correlate receptor regulatorychanges with analgesic tolerance It has been shown thatsustained morphine treatment of rats leading to analgesictolerance did not change the density or G-protein couplingof the surface mu-opioid receptors However significantintracellular changes including upregulation of the mu-sites and their cognate G-proteins were noted in the lightmembrane fraction [28] In addition we have described that14-methoxymetopon an extremely potent centrally actingmu-opioid specific analgesic with low tolerance physicaldependence and other side effects when given to rats eitheracutely or chronically did not change the binding or G-protein signaling of mu-opioid receptors in rat brain subcel-lular membranes [29] We have hypothesized that whereassurface opioid receptors and their cognate G-proteins medi-ate the acute effect of opioids intracellular events may play acrucial role in the long-term changes elicited by chronic drugexposure [28]

In the present work we have examined the new endo-morphin analogue ACHC-EM2 for its antinociceptiveeffect and receptor regulatory changes after acute andprolonged in vivo treatments The model system usedis based on subcellular fractionation of rat brains fol-lowed by ligand binding and functional measurementsDetailed characterization of the subcellular fractions bymarker enzymes electron microscopy and receptor bind-ing experiments has been reported [28ndash31] To assesschanges in the intracellular distribution of mu-opioid recep-tors subcellular fractionation was combined with radioli-gand binding measurements The degree of desensitization

was estimated by ligand-stimulated guanosine-51015840-O-(3-[120574[35S]hio)triphosphate [35S]GTP120574S functional assays It isconcluded that the regulatory changes accompanyingACHC-EM2 analgesic tolerance are distinct from those of theprototypic mu-agonist peptide DAMGO (Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol) and its chloromethyl derivative DAMCK(Tyr-D-Ala-Gly-(NMe)Phe-CH

2Cl)

2 Materials and Methods

21 Chemicals Endomorphin derivatives were synthesizedas published [3] DAMGO and [3H]DAMGO (36 Cimmol)were prepared and kindly provided to us by Drs Farkasand Toth (Biological Research Center Szeged Hungary)or purchased from Multiple Peptide System (San DiegoCA USA) DAMCK was synthesized as published [32] byAnna Magyar (ELTE Budapest Hungary) [35S]GTP120574S wasobtained from Isotope Institute Ltd (Budapest Hungary)Tris(hydroxymethyl)aminomethane (Tris free base)sodium chloride (NaCl) ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA) guanosine 51015840-diphosphate sodiumsalt (GDP) guanosine 51015840-triphosphate sodium salt (GTP)guanosine 51015840-[120574-thio]triphosphate tetralithium salt (GTP-120574-S-Li4) magnesium chloride hexahydrate (MgCl

2times 6H2O)

dithiothreitol sucrose and Nembutal were purchased fromSigma-Aldrich (St Louis MO USA) Bradford reagent wasfrom Bio-Rad Laboratories (Hercules CA USA)

22 Animals Wistar rats (250ndash500 g) were used They werekept under a standard light-dark cycle with food and wateravailable ad libitum The animals were kept and treatedaccording to the rules of the Ethical Committee for theProtection of Animals in Research (University of Szeged andBiological ResearchCenter of theHungarianAcademy of Sci-ences Szeged Hungary) All efforts were made to minimizetheir suffering during treatmentsThe animalswere randomlyassigned to treatment groups (119899 = 4dosetreatment)

23 Surgery For intracerebroventricular (icv) peptide admin-istration the rats were implanted with a stainless steelLuer cannula (10mm long) aimed at the right lateral cere-bral ventricle under Nembutal (35mgkg intraperitoneally)anesthesia [32] The stereotaxic coordinates were 02mmposterior 17mm lateral to the bregma and 37mm deepfrom the dural surface according to the atlas of Pellegrinoet al [33] Cannulas were secured to the skull with dentalcement and acrylate The experiments were started 5 daysafter icv cannulation All icv treatments were delivered tofreely moving animals to prevent unspecific effects Uponconclusion of the experiments 10 120583L of methylene blue wasinjected into the ventricle of decapitated animals and theposition of the cannula was inspected visually Animals withimproper cannula placement were excluded from the finalstatistical analysis

24 In Vivo Opioid Treatments The peptides were dissolvedin artificial cerebrospinal fluid (CSF) and injected in a volumeof 2 120583L Endomorphins were administered either acutely at





















0

10

20

30

40

50

60

10 15 20 40(min)

Ant

inoc

icep

tive e

ffect

()

Control100 pg10 ng

1120583g20120583g

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

(a)

10 15 20 400

10

20

30

40

50

60

Ant

inoc

icep

tive e

ffect

()

(min)

Control100 pg10 ng

1120583g20120583g

lowast

lowast

lowast

lowast

lowast

lowast

lowast

(b)


0

10

20

30

40

50

1 3 5 7 9Days

Ant

inoc

icep

tive e

ffect

()

lowast

lowast

lowast

(a)

0

10

20

30

40

50

1 3 5 7 9Days

Ant

inoc

icep

tive e

ffect

()

lowast

lowast

lowast lowast

(b)





3 Results














0

20

40

60

80

100

120

DAMGOEM-2ACHC-EM2

log[Ligand] (M)

Stim

ulat

ion

(ove

r bas

al) (

)






50values of






0

10

20

30

40

50

60lowast

[3H

] DA

MG

O b

indi

ng (

chan

ge)

(a)

0

20

40

60

minus40

minus20

lowast[3H

] DA

MG

O b

indi

ng (

chan

ge)

(b)

0

20

40

60

minus40

minus20

lowast

[3H

] DA

MG

O b

indi

ng (

chan

ge)

lowast

(c)








4 Discussion







50of DAMGO by about





5 Conclusions




Acknowledgments


References






















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





















0

10

20

30

40

50

60

10 15 20 40(min)

Ant

inoc

icep

tive e

ffect

()

Control100 pg10 ng

1120583g20120583g

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

(a)

10 15 20 400

10

20

30

40

50

60

Ant

inoc

icep

tive e

ffect

()

(min)

Control100 pg10 ng

1120583g20120583g

lowast

lowast

lowast

lowast

lowast

lowast

lowast

(b)


0

10

20

30

40

50

1 3 5 7 9Days

Ant

inoc

icep

tive e

ffect

()

lowast

lowast

lowast

(a)

0

10

20

30

40

50

1 3 5 7 9Days

Ant

inoc

icep

tive e

ffect

()

lowast

lowast

lowast lowast

(b)





3 Results














0

20

40

60

80

100

120

DAMGOEM-2ACHC-EM2

log[Ligand] (M)

Stim

ulat

ion

(ove

r bas

al) (

)






50values of






0

10

20

30

40

50

60lowast

[3H

] DA

MG

O b

indi

ng (

chan

ge)

(a)

0

20

40

60

minus40

minus20

lowast[3H

] DA

MG

O b

indi

ng (

chan

ge)

(b)

0

20

40

60

minus40

minus20

lowast

[3H

] DA

MG

O b

indi

ng (

chan

ge)

lowast

(c)








4 Discussion







50of DAMGO by about





5 Conclusions




Acknowledgments


References






















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of










0

20

40

60

80

100

120

DAMGOEM-2ACHC-EM2

log[Ligand] (M)

Stim

ulat

ion

(ove

r bas

al) (

)






50values of






0

10

20

30

40

50

60lowast

[3H

] DA

MG

O b

indi

ng (

chan

ge)

(a)

0

20

40

60

minus40

minus20

lowast[3H

] DA

MG

O b

indi

ng (

chan

ge)

(b)

0

20

40

60

minus40

minus20

lowast

[3H

] DA

MG

O b

indi

ng (

chan

ge)

lowast

(c)








4 Discussion







50of DAMGO by about





5 Conclusions




Acknowledgments


References






















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

10

20

30

40

50

60lowast

[3H

] DA

MG

O b

indi

ng (

chan

ge)

(a)

0

20

40

60

minus40

minus20

lowast[3H

] DA

MG

O b

indi

ng (

chan

ge)

(b)

0

20

40

60

minus40

minus20

lowast

[3H

] DA

MG

O b

indi

ng (

chan

ge)

lowast

(c)








4 Discussion







50of DAMGO by about





5 Conclusions




Acknowledgments


References






















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



50of DAMGO by about





5 Conclusions




Acknowledgments


References






















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

ligand-specific regulation of the endogenous mu-opioid receptor

Documents