Advertisement

A Common Human μ-Opioid Receptor Genetic Variant Diminishes the Receptor Signaling Efficacy in Brain Regions Processing the Sensory Information of Pain*

Open AccessPublished:December 30, 2008DOI:https://doi.org/10.1074/jbc.M807030200
      The single nucleotide polymorphism 118A>G of the human μ-opioid receptor gene OPRM1, which leads to an exchange of the amino acid asparagine (N) to aspartic acid (D) at position 40 of the extracellular receptor region, alters the in vivo effects of opioids to different degrees in pain-processing brain regions. The most pronounced N40D effects were found in brain regions involved in the sensory processing of pain intensity. Using the μ-opioid receptor-specific agonist DAMGO, we analyzed the μ-opioid receptor signaling, expression, and binding affinity in human brain tissue sampled postmortem from the secondary somatosensory area (SII) and from the ventral posterior part of the lateral thalamus, two regions involved in the sensory processing and transmission of nociceptive information. We show that the main effect of the N40D μ-opioid receptor variant is a reduction of the agonist-induced receptor signaling efficacy. In the SII region of homo- and heterozygous carriers of the variant 118G allele (n = 18), DAMGO was only 62% as efficient (p = 0.002) as in homozygous carriers of the wild-type 118A allele (n = 15). In contrast, the number of [3H]DAMGO binding sites was unaffected. Hence, the μ-opioid receptor G-protein coupling efficacy in SII of carriers of the 118G variant was only 58% as efficient as in homozygous carriers of the 118A allele (p < 0.001). The thalamus was unaffected by the OPRM1 118A>G SNP. In conclusion, we provide a molecular basis for the reduced clinical effects of opioid analgesics in carriers of μ-opioid receptor variant N40D.
      The human μ-opioid receptor variant N40D coded by the single nucleotide polymorphism (SNP)
      The abbreviations used are: SNP, single nucleotide polymorphism; ANOVA, analysis of variance; GTPγS, guanosine 5′-3-O-(thio)triphosphate; rm, repeated measures.
      118A>Gofthe μ-opioid receptor gene, OPRM1 (dbSNP rs1799971; allelic frequency 8.2-17%) has been found to be associated with diminished opioid effects in experimental (
      • Lötsch J.
      • Skarke C.
      • Grosch S.
      • Darimont J.
      • Schmidt H.
      • Geisslinger G.
      ,
      • Skarke C.
      • Darimont J.
      • Schmidt H.
      • Geisslinger G.
      • Lotsch J.
      ,
      • Romberg R.
      • Olofsen E.
      • Sarton E.
      • den Hartigh J.
      • Taschner P.E.
      • Dahan A.
      ,
      • Romberg R.R.
      • Olofsen E.
      • Bijl H.
      • Taschner P.E.
      • Teppema L.J.
      • Sarton E.Y.
      • van Kleef J.W.
      • Dahan A.
      ,
      • Oertel B.G.
      • Schmidt R.
      • Schneider A.
      • Geisslinger G.
      • Lotsch J.
      ) and clinical (
      • Klepstad P.
      • Rakvag T.T.
      • Kaasa S.
      • Holthe M.
      • Dale O.
      • Borchgrevink P.C.
      • Baar C.
      • Vikan T.
      • Krokan H.E.
      • Skorpen F.
      ,
      • Chou W.Y.
      • Wang C.H.
      • Liu P.H.
      • Liu C.C.
      • Tseng C.C.
      • Jawan B.
      ,
      • Campa D.
      • Gioia A.
      • Tomei A.
      • Poli P.
      • Barale R.
      ,
      • Sia A.T.
      • Lim Y.
      • Lim E.C.
      • Goh R.W.
      • Law H.Y.
      • Landau R.
      • Teo Y.Y.
      • Tan E.C.
      ) settings. However, in vitro experiments trying to elucidate the underlying molecular mechanisms provided inconsistent results, falling short to support the comparatively strong clinical evidence of decreased opioid effects in carriers of the variant 118G allele.
      Among the reported molecular consequences of the 118A>G polymorphism is a three times higher binding affinity of β-endorphin at the N40D μ-opioid receptors expressed in transfected Syrian hamster adenovirus-12-induced tumor cells (AV-12), whereas the affinity of other exogenous opioids was unaffected (
      • Bond C.
      • LaForge K.S.
      • Tian M.
      • Melia D.
      • Zhang S.
      • Borg L.
      • Gong J.
      • Schluger J.
      • Strong J.A.
      • Leal S.M.
      • Tischfield J.A.
      • Kreek M.J.
      • Yu L.
      ). In contrast, a leftward shift of the potencies of DAMGO and morphine-mediated Ca2+ channel inhibition has been shown in N40D variant μ-opioid receptor expressed in rat sympathetic superior cervical ganglion (SCG) neurons (
      • Margas W.
      • Zubkoff I.
      • Schuler H.G.
      • Janicki P.K.
      • Ruiz-Velasco V.
      ). However, both findings of a higher ligand affinity and potency were not reproduced in three attempts using N40D variant μ-opioid receptors transfected Cercopithecus aethiops kidney cells (COS), human 293 embryonic kidney cells (HEK293), and again AV-12 cells (
      • Befort K.
      • Filliol D.
      • Decaillot F.M.
      • Gaveriaux-Ruff C.
      • Hoehe M.R.
      • Kieffer B.L.
      ,
      • Beyer A.
      • Koch T.
      • Schroder H.
      • Schulz S.
      • Hollt V.
      ,
      • Kroslak T.
      • Laforge K.S.
      • Gianotti R.J.
      • Ho A.
      • Nielsen D.A.
      • Kreek M.J.
      ). Moreover, they do not explain the decreased clinical opioid potency. As an alternative mechanism, decreased μ-opioid receptor expression caused by the OPRM1 118G variant has been demonstrated in transfected HEK293 cells (
      • Beyer A.
      • Koch T.
      • Schroder H.
      • Schulz S.
      • Hollt V.
      ) and Chinese hamster ovary cells (CHO) (
      • Zhang Y.
      • Wang D.
      • Johnson A.D.
      • Papp A.C.
      • Sadee W.
      ). However, decreased μ-opioid receptor expression as the molecular mechanism for the 118A>G variant was contradicted by the report of unchanged receptor expression in N40D variant receptors expressing HEK293 cells (
      • Befort K.
      • Filliol D.
      • Decaillot F.M.
      • Gaveriaux-Ruff C.
      • Hoehe M.R.
      • Kieffer B.L.
      ). Adding to the inconsistency of molecular findings in transfected cell lines, a recent report demonstrated either decreased or unchanged receptor expression in HEK293 and AV-12 cells, respectively, depending on the transfection method (
      • Kroslak T.
      • Laforge K.S.
      • Gianotti R.J.
      • Ho A.
      • Nielsen D.A.
      • Kreek M.J.
      ). Finally, a single investigation assessing OPRM1 118A>G effects in postmortem human brain tissue indicated reduced OPRM1 mRNA levels in carriers of the variant 118G allele (
      • Zhang Y.
      • Wang D.
      • Johnson A.D.
      • Papp A.C.
      • Sadee W.
      ). However, the effects of the variant 118G allele on μ-opioid receptor protein expression and function were not examined in that investigation. Moreover, whether the analyzed pooled brain tissue from regions such as cortical lobes and pons are to be considered as primary parts of the pain matrix (
      • Apkarian A.V.
      • Bushnell M.C.
      • Treede R.D.
      • Zubieta J.K.
      ) is unknown.
      Thus, attempts to identify the molecular mechanism of the clinical functionality of the OPRM1 118A>G genetic variant have produced inconsistent or incomplete results that do not satisfyingly explain the clinically observed decreased analgesic effects of opioids in carriers of this variant. This is probably partly attributable to the artificial conditions in non-human non-neuronal cells and partly to the neglect of region-dependent effects on μ-opioidergic mechanisms in the human brain, which is strongly suggested to play a role for 118A>G by recent fMRI results (
      • Oertel B.G.
      • Preibisch C.
      • Wallenhorst T.
      • Hummel T.
      • Geisslinger G.
      • Lanfermann H.
      • Lotsch J.
      ). Specifically, the presence of the OPRM1 118A>G SNP decreased the analgesic effects of alfentanil primarily at brain regions processing the sensory dimension of pain whereas brain regions processing the affective dimension appeared to be unaffected. Considering this fMRI evidence for its brain region-specific effects, we analyzed by means of the μ-opioid receptor-specific agonist DAMGO the consequences of the OPRM1 118A>G SNP for μ-opioid receptor signaling, expression and ligand binding affinity in human brain tissue from brain regions known to take part in the processing of the sensory dimension of pain. This included the ventral posterior part of the lateral thalamus where the spinothalamic pathway transmitting peripheral noxious information from the dorsal horn of the spinal cord to the thalamus terminates (
      • Price D.D.
      ). Furthermore, it included the secondary somatosensory cortex (SII) as one of its cortical projection areas processing the sensory dimension of pain (
      • Coghill R.C.
      • Sang C.N.
      • Maisog J.M.
      • Iadarola M.J.
      ,
      • Bornhovd K.
      • Quante M.
      • Glauche V.
      • Bromm B.
      • Weiller C.
      • Buchel C.
      ) where opioid effects on pain-related brain activation have been shown to be affected by the OPRM1 118A>G SNP (
      • Oertel B.G.
      • Preibisch C.
      • Wallenhorst T.
      • Hummel T.
      • Geisslinger G.
      • Lanfermann H.
      • Lotsch J.
      ).

      EXPERIMENTAL PROCEDURES

      Tissue Collection and OPRM1 Genetic Testing-Human brain tissue was sampled during autopsy from the secondary somatosensory cortex SII and the ventral posterior part of the lateral thalamus from 95 subjects without known actual opioid treatment or actual or previous drug addiction (ethics approval obtained). DNA was extracted from the brain tissue using the BioRobot® EZ1 work station and the EZ1 DNA Tissue kit (Qiagen, Hilden, Germany). Subsequently, by means of a validated Pyrosequencing™ assay (
      • Skarke C.
      • Kirchhof A.
      • Geisslinger G.
      • Lotsch J.
      ), these samples were diagnosed to contain 68 non-carriers, 24 heterozygous, and three homozygous carriers of the variant OPRM1 118G allele, of which brain tissue was analyzed from all three homozygous carriers, from 18 heterozygous carriers, and from a random sample of 16 non-carriers of the variant drawn to match the number of carriers (for more subject-related details see supplemental Table S1). Because of limited tissue availability, it was not possible to employ every subject in all experiments. Therefore, the exact sample sizes are indicated at the respective results. Experiments were performed in an investigator-blinded fashion with respect to the sample genotype.
      Membrane Preparation-Tissue (1-1.5 g) was homogenized with a fast rotating Ultra-Turrax (Janke & Kunkel GmbH, Staufen i. Breisgau, Germany) in 10-15 volumes of homogenization buffer (10 mm Tris-HCl, pH 7.4, 290 mm d(+)-saccharose). The crude tissue lysate was immediately centrifuged at 1,000 × g at 4 °C for 15 min, and the supernatant again at 45,000 × g, at 4 °C for 45 min. The pellet was resuspended in ice-cold isolation buffer (50 mm Tris-HCl, pH 7.4) and stored at -80 °C pending further analysis. Protein concentrations were measured according to the method of Bradford (
      • Bradford M.M.
      ).
      Assessment of DAMGO-induced μ-Opioid Receptor Signaling Efficacy and Potency-The efficacy (BNet) and potency (EC50) of d-Ala2,N-MePhe4,Gly-ol5-enkephalin (DAMGO, Sigma-Aldrich) to trigger μ-opioid receptor signaling were assessed by fitting a sigmoidal concentration-response curve to [35S]GTPγS binding data (for experimental details and equations see supplemental material). The experiments were performed as published previously (
      • Gonzalez-Maeso J.
      • Rodriguez-Puertas R.
      • Gabilondo A.M.
      • Meana J.J.
      ). [35S]GTPγS binding data were acquired by incubation of membrane proteins with 0.5 nm [35S]GTPγS (Amersham Biosciences, Uppsala, Sweden) in the presence of 50 μm GDP (Sigma-Aldrich) and increasing concentrations of DAMGO (0 nm-100 μm). In addition, control experiments were performed under equal conditions with the non-opioid agonist adenosine (0 nm to 100 μm; Sigma-Aldrich) to verify that observed differences between genotype groups were not related to potential confounders, e.g. tissue variability.
      Assessment of [3H]DAMGO Binding Sites and Affinity-The number of [3H]DAMGO binding sites (BMax) and its affinity (KD) were assessed by fitting a one site hyperbola to the data of the specific saturation binding of [3H]DAMGO (Amersham Biosciences). Specific [3H]DAMGO binding data were acquired by incubation of membrane proteins with at least 10 different concentrations of [3H]DAMGO in the range of 0.07-14.42 nm in the presence and absence of 10 μm naloxone (Naloxon-Curamed, Curamed, Karlsruhe, Germany). Control experiments were performed by incubation of membrane proteins with at least 6 different concentrations of [3H]adenosine (Amersham Biosciences) in the range of 17-500 nm in the presence and absence of 1 mm adenosine (Sigma-Aldrich).
      Furthermore, OPRM1 mRNA expression was analyzed by means of quantitative real-time PCR (RT-PCR) analysis of OPRM1 mRNA expression in relation to human ACTB (β-actin) mRNA (for experimental details and equations, see supplemental materials).
      Statistics-The numerical values of the BNet, EC50, BMax, and KD were obtained by non-linear regression analyzes of individual data using GraphPad Prism® 5.01 (GraphPad Software, San Diego, CA). OPRM1 mRNA expression was determined by using the comparative Ct method (2-ΔΔCt; for equations see supplemental materials). Because BNet(DAMGO) correlated statistically significantly with BMax([3H]DAMGO) in SII (Pearson correlation: 0.41, p = 0.018) and thalamus (0.61, p < 0.001), the net effect of agonist binding per μ-opioid receptor was calculated separately for each subject and each brain region as the quotient of DAMGO efficacy and the number of DAMGO binding sites, BNet(DAMGO)/BMax([3H]DAMGO). This allowed for comparison of μ-opioid receptor-G-protein coupling efficacy between brain regions and genotype groups (
      • Sim L.J.
      • Selley D.E.
      • Xiao R.
      • Childers S.R.
      ,
      • Maher C.E.
      • Selley D.E.
      • Childers S.R.
      ,
      • Ko M.C.
      • Lee H.
      • Harrison C.
      • Clark M.J.
      • Song H.F.
      • Naughton N.N.
      • Woods J.H.
      • Traynor J.R.
      ). Similarly, the BNet(adenosine)/BMax([3H]adenosine) was calculated for the control experiments. Because of the small number of homozygous 118G carriers, hetero- and homozygous carriers of the 118G allele were pooled and subsequent group comparisons were made for two groups (i.e. non-carriers of the 118G allele “118AA” and carriers of the “118G” allele “118G”). The region-specific effects of the OPRM1 118A>G single nucleotide polymorphism were analyzed by means of multivariate analysis of variance for repeated measures (rm-ANOVA), with region (i.e. SII or thalamus; degrees of freedom, df = 1) as within-subject factor and 118A>G (i.e. 118AA or 118G; df = 1) as between-subject factor (SPSS 15.0.1.1, SPSS Inc., Chicago, IL). The α-level was set at 0.05. Post-hoc comparisons for statistical main effects were done by t-tests. In the case of significant effects of the ORPM1 118A>G genotype, the influence of potential confounders was checked, e.g. the subject′s sex (t-tests), age at death, postmortem delay, and tissue storage period (correlation analyses).

      RESULTS

      Subject-related Data-The group of homozygous carriers of the wild-type allele (118AA) and the group of hetero- and homozygous carriers of the variant 118G allele (118G) differed not with respect to the subjects' sex (118AA: 6 men and 10 women; 118G: 10 men and 11 women; Chi2-test: p = 0.74), age at death (118AA: 53.2 years (mean), 19-84 years (range); 118G: 57.8 years, 23-92 years; Independent samples t-tests: p = 0.50), postmortem delay (118AA: 31.7 h, 4-85 h; 118G: 31.1 h, 7-85 h; p = 0.94), and tissue storage period (118AA: 18.9 months, 10-28 months; 118G: 17.3 months, 9-28 months; p = 0.39).
      DAMGO-induced μ-Opioid Receptor Signaling Efficacy and Potency-The efficacy of DAMGO to stimulate [35S]GTPγS binding, BNet(DAMGO), was similar in the SII and thalamus as indicated by the absence of a statistical main effect of rm-ANOVA factor region (F (1,1,31) = 0.003, p = 0.96; Fig. 1 and Table 1). Similarly, the 118G allele had no effect on the efficacy of DAMGO as indicated by the absence of a statistical main effect of rm-ANOVA factor 118A>G (F (1,1,31) = 1.78, p = 0.19). However, the genotype influenced significantly the DAMGO efficacy in a brain region-dependent manner (rm-ANOVA interaction region by 118A>G: F (1,1,31) = 6.78, p = 0.014). Specifically, a genotype effect was seen exclusively in SII, where in hetero- and homozygous 118G carriers DAMGO was only 62% as efficient as in homozygous carriers of the 118A allele (t test: t(31) = 3.45, p = 0.002). This was not the case for the thalamus, where the DAMGO efficacy was not different between non-carriers and carriers of the variant 118G allele (t test: t(31) = 0.39, p = 0.70). The potential confounders sex (t test: t(31) = -1.30, p = 0.20), age at death, postmortem delay, and storage period (Pearson correlation: p > 0.08) had no influence on BNet(DAMGO) in SII.
      Figure thumbnail gr1
      FIGURE 1Effects of the OPRM1 118A>G SNP on the agonist-induced receptor signaling (left figures), the agonist binding (middle figures), and the receptor-G-protein coupling efficacies (right figures) of DAMGO and adenosine in the secondary somatosensory area, SII, and the ventral posterior part of the lateral thalamus. The [35S]GTPγS binding data showed that the efficacy of DAMGO to stimulate the [35S]GTPγS binding was significantly reduced in the SII region of carriers of the variant 118G allele compared with non-carriers (1st row, left figure; nonlinear fit to mean of data/solid and dashed lines, 95% CI/bars). The saturation binding experiments indicated that the different DAMGO efficacies were not related to different amounts of μ-opioid receptor binding sites (1st row, middle figure; nonlinear fit to mean of data/solid and dashed lines, 95% CI/bars). This is confirmed by the higher ratio of BNet(DAMGO)/BMax([3H]DAMGO) in the SII region of carriers of the variant 118G allele compared with non-carriers (1st row, right figure; median/solid line, mean/dotted line, min and max/bars). The absence of OPRM1 118A>G SNP effects on the control experiments with the non-opioid agonist adenosine in the SII region verified that the observed differences of the DAMGO efficacy between genotype groups were not related to μ-opioid receptor independent factors like tissue variability (2nd row). In the thalamus, the OPRM1 118A>G SNP neither affected the agonist-induced receptor signaling, receptor binding sites, and receptor-G-protein coupling efficacies of DAMGO (3rd row) nor of adenosine (4th row).
      TABLE 1[35S]GTPγS binding data after DAMGO and adenosine stimulation in tissue samples from the secondary somatosensory area, SII, and from the ventral posterior part of the lateral thalamus
      AgonistValueNOPRM1SIIThalamusUnit
      118Value95% CIValue95% CI
      DAMGOBNet
      BNet, maximum net binding of [35S]GTPγS after agonist stimulation. The genotype influenced the DAMGO efficacy in a significant brain region-dependent manner (rm-ANOVA interaction region by 118A>G: F (1,1,31) = 6.78, p = 0.014). Specifically, in the SII of 118G carriers, DAMGO was only 62% as efficient as in homozygous carriers of the 118A allele (t-test: t(31) = 3.45, p = 0.002). In contrast, the DAMGO efficacy in the thalamus was not significantly different between non-carriers and carriers of the variant 118G allele of (t-test: t(31) = 0.39, p = 0.70).
      15AA283[236,330]221[148,295]fmol/mg protein
      18
      Consisting of 15 heterozygous carriers (118AG) and 3 homozygous carriers (118GG) of the 118G allele.
      G176[133,219]240[173,308]
      EC50
      EC50, concentration of agonist producing 50% of the maximum net binding of [35S]GTPγS.
      15AA1.27[0.65,1.89]0.26[0,0.55]μm
      18
      Consisting of 15 heterozygous carriers (118AG) and 3 homozygous carriers (118GG) of the 118G allele.
      G0.94[0.38,1.51]0.39[0.13,0.65]
      AdenosineBNet13AA373[306,440]432[338,527]fmol/mg protein
      17
      Consisting of 14 heterozygous carriers (118AG) and 3 homozygous carriers (118GG) of the 118G allele.
      G345[286,403]360[277,442]
      EC5013AA1.17[0.49,1.85]1.66[0.73,2.58]μm
      17
      Consisting of 14 heterozygous carriers (118AG) and 3 homozygous carriers (118GG) of the 118G allele.
      G0.94[0.35,1.53]1.44[0.63,2.24]
      a BNet, maximum net binding of [35S]GTPγS after agonist stimulation. The genotype influenced the DAMGO efficacy in a significant brain region-dependent manner (rm-ANOVA interaction region by 118A>G: F (1,1,31) = 6.78, p = 0.014). Specifically, in the SII of 118G carriers, DAMGO was only 62% as efficient as in homozygous carriers of the 118A allele (t-test: t(31) = 3.45, p = 0.002). In contrast, the DAMGO efficacy in the thalamus was not significantly different between non-carriers and carriers of the variant 118G allele of (t-test: t(31) = 0.39, p = 0.70).
      b Consisting of 15 heterozygous carriers (118AG) and 3 homozygous carriers (118GG) of the 118G allele.
      c EC50, concentration of agonist producing 50% of the maximum net binding of [35S]GTPγS.
      d Consisting of 14 heterozygous carriers (118AG) and 3 homozygous carriers (118GG) of the 118G allele.
      Similar to DAMGO-stimulated [35S]GTPγS binding, the control experiments with the non-opioid agonist adenosine (Fig. 1 and Table 1) showed that the efficacy of adenosine, BNet(adenosine), was similar in both brain regions (rm-ANOVA factor region: F (1,1,28) = 1.46, p = 0.24) and that it was unaffected by the 118G allele (rm-ANOVA factor 118A>G: F (1,1,28) = 1.36, p = 0.25). However, in contrast to DAMGO-stimulated [35S]GTPγS binding, the genotype had no significant influence on the adenosine efficacy in a brain region-dependent manner (interaction region by 118A>G: F (1,1,28) = 0.52, p = 0.48). This further indicates that the observed brain region-dependent differences of [35S]GTPγS binding after DAMGO stimulation between non-carriers and carriers of the 118G allele were indeed related to the 118G allele, and not the result of potential confounders, e.g. tissue variability.
      The potency of DAMGO to stimulate [35S]GTPγS binding, EC50(DAMGO), was brain region-dependent with a significantly lower potency of DAMGO in SII than in the thalamus as indicated by the higher EC50(DAMGO) in SII (rm-ANOVA factor region: F (1,1,31) = 13.77, p = 0.001). However, the potency did not differ significantly between homozygous carriers of the 118A allele and hetero- and homozygous carriers of the 118G allele (p ≥ 0.28; Table 1). In contrast, the control agonist adenosine was equally potent in SII and thalamus of carriers of either genotype as indicated by the absence of any statistically significant main effect of the rm-ANOVA (p ≥ 0.23; Table 1).
      [3H]DAMGO Binding Sites and Affinity-The number of DAMGO binding sites, BMax([3H]DAMGO), was significantly lower in SII than in the thalamus (rm-ANOVA factor region: F (1,1,31) = 16.28, p < 0.001; Fig. 1 and Table 2). The lower number of DAMGO binding sites was affirmed by lower OPRM1 mRNA expression in SII compared with the thalamus (rm-ANOVA factor region: F (1,1,20) = 7.03, p = 0.015). The OPRM1 118A>G SNP had no statistically significant effect on the number of DAMGO binding sites (p ≥ 0.46) and OPRM1 mRNA-expression levels (p ≥ 0.60), respectively.
      TABLE 2[3H]DAMGO binding data, [3H]adenosine binding data, and OPRM1 mRNA expression data in tissue samples from the secondary somatosensory area, SII, and from the ventral posterior part of the lateral thalamus
      AgonistValueNOPRM1SIIThalamusUnit
      118Value95% CIValue95% CI
      [3H]DAMGOBMax
      BMax, maximum number of agonist binding sites.
      15AA97[71,123]151[115,186]fmol/mg protein
      18
      Consisting of 15 heterozygous carriers (118AG) and 3 homozygous carriers (118GG) of the 118G allele.
      G114[91,138]151[118,183]
      KD
      KD, equilibrium dissociation constant.
      15AA1.9[1.3,2.5]1.4[0.8,2.0]nm
      18
      Consisting of 15 heterozygous carriers (118AG) and 3 homozygous carriers (118GG) of the 118G allele.
      G2.5[1.9,3.1]1.8[1.2,2.3]
      [3H]AdenosineBMax12AA10.0[6.6,13.5]13.0[7.7,18.4]pmol/mg protein
      17
      Consisting of 14 heterozygous carriers (118AG) and 3 homozygous carriers (118GG) of the 118G allele.
      G12.4[9.5,15.3]15.7[11.2,20.2]
      KD12AA696[194,1198]505[0,1134]nm
      17
      Consisting of 14 heterozygous carriers (118AG) and 3 homozygous carriers (118GG) of the 118G allele.
      G353[55,652]348[52,643]
      OPRM1 mRNA2−ΔΔCt
      2−ΔΔCt, OPRM1 mRNA expression in relation to human ACTB (β-actin) mRNA.
      11AA1.5[0.7,2.4]3.0[1.1,4.8]-
      11
      Consisting of 8 heterozygous carriers (118AG) and 3 homozygous carriers (118GG) of the 118G allele.
      G1.4[0.6,2.2]3.6[1.7,5.4]
      a BMax, maximum number of agonist binding sites.
      b Consisting of 15 heterozygous carriers (118AG) and 3 homozygous carriers (118GG) of the 118G allele.
      c KD, equilibrium dissociation constant.
      d Consisting of 14 heterozygous carriers (118AG) and 3 homozygous carriers (118GG) of the 118G allele.
      e 2−ΔΔCt, OPRM1 mRNA expression in relation to human ACTB (β-actin) mRNA.
      f Consisting of 8 heterozygous carriers (118AG) and 3 homozygous carriers (118GG) of the 118G allele.
      The affinity of [3H]DAMGO at the μ-opioid receptors in SII was significantly lower than in the thalamus as indicated by the higher KD([3H]DAMGO) in SII (rm-ANOVA factor region: F (1,1,31) = 8.30, p = 0.007). Again, the OPRM1 118A>G SNP had no statistically significant effect on the affinity of [3H]DAMGO (p ≥ 0.16; Fig. 1 and Table 2).
      BMax and KD of [3H]adenosine were similar in both brain regions and unaffected by the OPRM1 118A>G SNP as indicated by the absence of a statistically significant main effect of the rm-ANOVA (p ≥ 0.08; Table 2).
      μ-Opioid Receptor G-protein Coupling Efficacy-Analysis of the ratio BNet(DAMGO)/BMax([3H]DAMGO) confirmed a brain region-dependent μ-opioid receptor G-protein coupling efficacy (rm-ANOVA factor region: F (1,1,31) = 30.81, p < 0.001; Fig. 1 and Table 3), which was modulated by the 118A>G genotype (rm-ANOVA factor 118A>G: F (1,1,31) = 8.42, p = 0.007 and rm-ANOVA interaction region by 118A>G: F (1,1,31) = 19.05, p < 0.001). Post-hoc comparisons showed that BNet(DAMGO)/BMax([3H]DAMGO) in SII of 118G carriers was only 58% as efficient as in 118AA carriers (t test: t(31) = 4.31, p < 0.001) while it was similar in the thalamus of 118AA and 118G carriers (t test: t(31) = 0.23, p = 0.82). The potential confounders sex (t test: t(31) = -0.39, p = 0.70), age at death, postmortem delay, and storage period (Pearson correlation: p > 0.09) had no influence on BNet(DAMGO)/BMax([3H]DAMGO) in SII.
      TABLE 3Quotient of the maximum net binding of [35S]GTPγS after agonist stimulation and maximum number of agonist binding sites in tissue samples from the secondary somatosensory area, SII, and from the ventral posterior part of the lateral thalamus
      ValueNOPRM1SIIThalamus
      118Value95% CIValue95% CI
      BNet (DAMGO)/BMax ([3H]DAMGO)
      BNet/BMax, maximum net binding of [35S]GTPγS per receptor after agonist stimulation. Analysis of BNet(DAMGO)/BMax([3H]DAMGO) showed a region-dependent μ-opioid receptor G-protein coupling efficacy (rm-ANOVA factor region: F (1,1,31) = 30.81, p < 0.001), which was modulated by the genotype (rm-ANOVA factor 118A>G: F (1,1,31) = 8.42, p < 0.001 and rm-ANOVA interaction region by 118A>G: F (1,1,31) = 19.05, p < 0.001). Post-hoc comparisons showed that BNet (DAMGO)/BMax([3H]DAMGO) in SII of 118G carriers was only 58% as efficient as in 118AA carriers (t-test: t(31) = 4.31, p < 0.001) while it was similar in the thalamus of 118AA and 118G carriers (t-test: t(31) = 0.23, p = 0.82).
      15AA3.1[2.6,3.5]1.6[1.3,2.0]
      18
      Consisting of 15 heterozygous carriers (118AG) and 3 homozygous carriers (118GG) of the 118G allele.
      G1.8[1.3,2.2]1.6[1.2,1.9]
      BNet(adenosine)/BMax([3H]adenosine)12AA0.036[0.026,0.045]0.047[0.031,0.064]
      17
      Consisting of 14 heterozygous carriers (118AG) and 3 homozygous carriers (118GG) of the 118G allele.
      G0.033[0.025,0.041]0.031[0.17,0.045]
      a BNet/BMax, maximum net binding of [35S]GTPγS per receptor after agonist stimulation. Analysis of BNet(DAMGO)/BMax([3H]DAMGO) showed a region-dependent μ-opioid receptor G-protein coupling efficacy (rm-ANOVA factor region: F (1,1,31) = 30.81, p < 0.001), which was modulated by the genotype (rm-ANOVA factor 118A>G: F (1,1,31) = 8.42, p < 0.001 and rm-ANOVA interaction region by 118A>G: F (1,1,31) = 19.05, p < 0.001). Post-hoc comparisons showed that BNet (DAMGO)/BMax([3H]DAMGO) in SII of 118G carriers was only 58% as efficient as in 118AA carriers (t-test: t(31) = 4.31, p < 0.001) while it was similar in the thalamus of 118AA and 118G carriers (t-test: t(31) = 0.23, p = 0.82).
      b Consisting of 15 heterozygous carriers (118AG) and 3 homozygous carriers (118GG) of the 118G allele.
      c Consisting of 14 heterozygous carriers (118AG) and 3 homozygous carriers (118GG) of the 118G allele.
      The ratio of the control experiments, BNet(adenosine)/BMax([3H]adenosine), was similar in SII and thalamus and, as expected, unaffected by the OPRM1 118A>G SNP (rm-ANOVA p ≥ 0.17; Table 3).

      DISCUSSION

      We show that the main functional effect of the OPRM1 118A>G variant is a decrease in the μ-opioid receptor signaling efficacy in a brain region, SII, known to be an important part of the pain matrix that codes for the pain intensity. Of the so far accumulated knowledge on the molecular mechanism underlying the functional consequences of the variant (
      • Bond C.
      • LaForge K.S.
      • Tian M.
      • Melia D.
      • Zhang S.
      • Borg L.
      • Gong J.
      • Schluger J.
      • Strong J.A.
      • Leal S.M.
      • Tischfield J.A.
      • Kreek M.J.
      • Yu L.
      ,
      • Margas W.
      • Zubkoff I.
      • Schuler H.G.
      • Janicki P.K.
      • Ruiz-Velasco V.
      ,
      • Befort K.
      • Filliol D.
      • Decaillot F.M.
      • Gaveriaux-Ruff C.
      • Hoehe M.R.
      • Kieffer B.L.
      ,
      • Beyer A.
      • Koch T.
      • Schroder H.
      • Schulz S.
      • Hollt V.
      ,
      • Kroslak T.
      • Laforge K.S.
      • Gianotti R.J.
      • Ho A.
      • Nielsen D.A.
      • Kreek M.J.
      ,
      • Zhang Y.
      • Wang D.
      • Johnson A.D.
      • Papp A.C.
      • Sadee W.
      ), the presented results agree best with the consistent experimental and clinical observations that this variant mainly decreases the opioid effects and/or increases the opioid dosing requirements to achieve analgesia (
      • Lötsch J.
      • Skarke C.
      • Grosch S.
      • Darimont J.
      • Schmidt H.
      • Geisslinger G.
      ,
      • Skarke C.
      • Darimont J.
      • Schmidt H.
      • Geisslinger G.
      • Lotsch J.
      ,
      • Romberg R.
      • Olofsen E.
      • Sarton E.
      • den Hartigh J.
      • Taschner P.E.
      • Dahan A.
      ,
      • Romberg R.R.
      • Olofsen E.
      • Bijl H.
      • Taschner P.E.
      • Teppema L.J.
      • Sarton E.Y.
      • van Kleef J.W.
      • Dahan A.
      ,
      • Oertel B.G.
      • Schmidt R.
      • Schneider A.
      • Geisslinger G.
      • Lotsch J.
      ,
      • Klepstad P.
      • Rakvag T.T.
      • Kaasa S.
      • Holthe M.
      • Dale O.
      • Borchgrevink P.C.
      • Baar C.
      • Vikan T.
      • Krokan H.E.
      • Skorpen F.
      ,
      • Chou W.Y.
      • Wang C.H.
      • Liu P.H.
      • Liu C.C.
      • Tseng C.C.
      • Jawan B.
      ,
      • Campa D.
      • Gioia A.
      • Tomei A.
      • Poli P.
      • Barale R.
      ,
      • Sia A.T.
      • Lim Y.
      • Lim E.C.
      • Goh R.W.
      • Law H.Y.
      • Landau R.
      • Teo Y.Y.
      • Tan E.C.
      ). The results are also in strong agreement with the fMRI observation that the genetic variant mainly affects the effects of opioid analgesics on the sensory component of pain (
      • Oertel B.G.
      • Preibisch C.
      • Wallenhorst T.
      • Hummel T.
      • Geisslinger G.
      • Lanfermann H.
      • Lotsch J.
      ), which is emphasized by the fact that the main molecular effect was presently found in the SII region. Based on the absent effect of the OPRM1 genotype in the control experiments with the non-opioid agonist adenosine and on the lack of influences of potential confounders, a contribution of factors such as tissue degradation to the differences of μ-opioid receptor signaling/-coupling between non-carriers and carriers of OPRM1 variant 118G appears to be unlikely.
      The present selection of brain regions was limited to SII and the thalamus, two brain regions known to be involved in the sensory processing of pain intensity. However, other brain regions as the two here selected may also be affected. This is suggested by the fMRI findings of 118G effects (
      • Oertel B.G.
      • Preibisch C.
      • Wallenhorst T.
      • Hummel T.
      • Geisslinger G.
      • Lanfermann H.
      • Lotsch J.
      ) being present also in other brain regions involved in the processing of pain, e.g. the posterior insula and SI. Although the μ-opioid receptor expression differed significantly between the two presently chosen brain regions, we could not reproduce a genotype difference in receptor expression levels as previously shown in human brain samples from pons and cortical lobes (
      • Zhang Y.
      • Wang D.
      • Johnson A.D.
      • Papp A.C.
      • Sadee W.
      ) and in OPRM1 118G-transfected non-brain or non-human cell lines (
      • Beyer A.
      • Koch T.
      • Schroder H.
      • Schulz S.
      • Hollt V.
      ,
      • Kroslak T.
      • Laforge K.S.
      • Gianotti R.J.
      • Ho A.
      • Nielsen D.A.
      • Kreek M.J.
      ,
      • Zhang Y.
      • Wang D.
      • Johnson A.D.
      • Papp A.C.
      • Sadee W.
      ), respectively. While it is possible that we missed an OPRM1 118G effect on μ-opioid receptor protein expression because [3H]DAMGO binds only to agonist states of the μ-opioid receptor (
      • Neilan C.L.
      • Janvey A.J.
      • Bolan E.
      • Berezowska I.
      • Nguyen T.M.
      • Schiller P.W.
      • Pasternak G.W.
      ), the analysis of OPRM1 mRNA expression affirms the conclusion that μ-opioid receptor expression is unaffected by the OPRM1 118A>G SNP in both investigated brain regions. However, the fact that we missed an OPRM1 118G effect on μ-opioid receptor expression may also relate to brain region specific effects of the 118G allele. Regionally limited alterations of μ-opioid receptor expression have already been shown for other SNPs known to affect the opioid system, e.g. the COMT 472G>A polymorphism (
      • Zubieta J.K.
      • Heitzeg M.M.
      • Smith Y.R.
      • Bueller J.A.
      • Xu K.
      • Xu Y.
      • Koeppe R.A.
      • Stohler C.S.
      • Goldman D.
      ,
      • Berthele A.
      • Platzer S.
      • Jochim B.
      • Boecker H.
      • Buettner A.
      • Conrad B.
      • Riemenschneider M.
      • Toelle T.R.
      ). Nevertheless, the presented results make a global reduction of receptor expression unlikely as a general molecular mechanism for the reduced analgesia in carriers of OPRM1 variant 118G.
      We would have probably missed a genotype effect if we had analyzed only a single brain region. The importance of brain region-dependent variable μ-opioid receptor expression (
      • Baumgartner U.
      • Buchholz H.G.
      • Bellosevich A.
      • Magerl W.
      • Siessmeier T.
      • Rolke R.
      • Hohnemann S.
      • Piel M.
      • Rosch F.
      • Wester H.J.
      • Henriksen G.
      • Stoeter P.
      • Bartenstein P.
      • Treede R.D.
      • Schreckenberger M.
      ,
      • Pilapil C.
      • Welner S.
      • Magnan J.
      • Gauthier S.
      • Quirion R.
      ) and function (
      • Maher C.E.
      • Selley D.E.
      • Childers S.R.
      ,
      • Rodriguez-Puertas R.
      • Gonzalez-Maeso J.
      • Meana J.J.
      • Pazos A.
      ) for the functional consequences of the 118A>G variant might be a major reason for the failed or contradictory results of previous investigations in cell cultures (
      • Bond C.
      • LaForge K.S.
      • Tian M.
      • Melia D.
      • Zhang S.
      • Borg L.
      • Gong J.
      • Schluger J.
      • Strong J.A.
      • Leal S.M.
      • Tischfield J.A.
      • Kreek M.J.
      • Yu L.
      ,
      • Margas W.
      • Zubkoff I.
      • Schuler H.G.
      • Janicki P.K.
      • Ruiz-Velasco V.
      ,
      • Befort K.
      • Filliol D.
      • Decaillot F.M.
      • Gaveriaux-Ruff C.
      • Hoehe M.R.
      • Kieffer B.L.
      ,
      • Beyer A.
      • Koch T.
      • Schroder H.
      • Schulz S.
      • Hollt V.
      ,
      • Kroslak T.
      • Laforge K.S.
      • Gianotti R.J.
      • Ho A.
      • Nielsen D.A.
      • Kreek M.J.
      ) that contrast with the consistent clinical findings of decreased opioid effects in carriers of variant 118G (
      • Lötsch J.
      • Skarke C.
      • Grosch S.
      • Darimont J.
      • Schmidt H.
      • Geisslinger G.
      ,
      • Skarke C.
      • Darimont J.
      • Schmidt H.
      • Geisslinger G.
      • Lotsch J.
      ,
      • Romberg R.
      • Olofsen E.
      • Sarton E.
      • den Hartigh J.
      • Taschner P.E.
      • Dahan A.
      ,
      • Romberg R.R.
      • Olofsen E.
      • Bijl H.
      • Taschner P.E.
      • Teppema L.J.
      • Sarton E.Y.
      • van Kleef J.W.
      • Dahan A.
      ,
      • Oertel B.G.
      • Schmidt R.
      • Schneider A.
      • Geisslinger G.
      • Lotsch J.
      ,
      • Klepstad P.
      • Rakvag T.T.
      • Kaasa S.
      • Holthe M.
      • Dale O.
      • Borchgrevink P.C.
      • Baar C.
      • Vikan T.
      • Krokan H.E.
      • Skorpen F.
      ,
      • Chou W.Y.
      • Wang C.H.
      • Liu P.H.
      • Liu C.C.
      • Tseng C.C.
      • Jawan B.
      ,
      • Campa D.
      • Gioia A.
      • Tomei A.
      • Poli P.
      • Barale R.
      ,
      • Sia A.T.
      • Lim Y.
      • Lim E.C.
      • Goh R.W.
      • Law H.Y.
      • Landau R.
      • Teo Y.Y.
      • Tan E.C.
      ). To which extent region-specific expression of μ-opioid receptor splice variants (
      • Pan Y.X.
      • Xu J.
      • Mahurter L.
      • Xu M.
      • Gilbert A.K.
      • Pasternak G.W.
      ,
      • Pan L.
      • Xu J.
      • Yu R.
      • Xu M.M.
      • Pan Y.X.
      • Pasternak G.W.
      ) and coupling to Gα and Gβγ subunits (
      • Chakrabarti S.
      • Prather P.L.
      • Yu L.
      • Law P.Y.
      • Loh H.H.
      ,
      • Kleuss C.
      • Scherubl H.
      • Hescheler J.
      • Schultz G.
      • Wittig B.
      ), which are known to influence the μ-opioid receptor signaling, contribute to the brain region-dependent effects of the N40D variant, may be addressed in the future.
      In conclusion, we provide a molecular basis for the reduced clinical effects of opioid analgesics in carriers of the μ-opioid receptor variant N40D. The variant has region-specific effects on the μ-opioid receptor signaling evident in parts of the pain matrix, consisting of a decreased receptor signaling efficiency after stimulation by exogenous opioid receptor agonists in a brain region known to process the sensory component of pain, i.e. pain intensity.

      Supplementary Material

      References

        • Lötsch J.
        • Skarke C.
        • Grosch S.
        • Darimont J.
        • Schmidt H.
        • Geisslinger G.
        Pharmacogenetics. 2002; 12: 3-9
        • Skarke C.
        • Darimont J.
        • Schmidt H.
        • Geisslinger G.
        • Lotsch J.
        Clin. Pharmacol. Ther. 2003; 73: 107-121
        • Romberg R.
        • Olofsen E.
        • Sarton E.
        • den Hartigh J.
        • Taschner P.E.
        • Dahan A.
        Anesthesiology. 2004; 100: 120-133
        • Romberg R.R.
        • Olofsen E.
        • Bijl H.
        • Taschner P.E.
        • Teppema L.J.
        • Sarton E.Y.
        • van Kleef J.W.
        • Dahan A.
        Anesthesiology. 2005; 102: 522-530
        • Oertel B.G.
        • Schmidt R.
        • Schneider A.
        • Geisslinger G.
        • Lotsch J.
        Pharmacogenet Genomics. 2006; 16: 625-636
        • Klepstad P.
        • Rakvag T.T.
        • Kaasa S.
        • Holthe M.
        • Dale O.
        • Borchgrevink P.C.
        • Baar C.
        • Vikan T.
        • Krokan H.E.
        • Skorpen F.
        Acta Anaesthesiol. Scand. 2004; 48: 1232-1239
        • Chou W.Y.
        • Wang C.H.
        • Liu P.H.
        • Liu C.C.
        • Tseng C.C.
        • Jawan B.
        Anesthesiology. 2006; 105: 334-337
        • Campa D.
        • Gioia A.
        • Tomei A.
        • Poli P.
        • Barale R.
        Clin. Pharmacol. Ther. 2008; 83: 559-566
        • Sia A.T.
        • Lim Y.
        • Lim E.C.
        • Goh R.W.
        • Law H.Y.
        • Landau R.
        • Teo Y.Y.
        • Tan E.C.
        Anesthesiology. 2008; 109: 520-526
        • Bond C.
        • LaForge K.S.
        • Tian M.
        • Melia D.
        • Zhang S.
        • Borg L.
        • Gong J.
        • Schluger J.
        • Strong J.A.
        • Leal S.M.
        • Tischfield J.A.
        • Kreek M.J.
        • Yu L.
        Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9608-9613
        • Margas W.
        • Zubkoff I.
        • Schuler H.G.
        • Janicki P.K.
        • Ruiz-Velasco V.
        J Neurophysiol. 2007; 97: 1058-1067
        • Befort K.
        • Filliol D.
        • Decaillot F.M.
        • Gaveriaux-Ruff C.
        • Hoehe M.R.
        • Kieffer B.L.
        J. Biol. Chem. 2001; 276: 3130-3137
        • Beyer A.
        • Koch T.
        • Schroder H.
        • Schulz S.
        • Hollt V.
        J. Neurochem. 2004; 89: 553-560
        • Kroslak T.
        • Laforge K.S.
        • Gianotti R.J.
        • Ho A.
        • Nielsen D.A.
        • Kreek M.J.
        J. Neurochem. 2007; 103: 77-87
        • Zhang Y.
        • Wang D.
        • Johnson A.D.
        • Papp A.C.
        • Sadee W.
        J. Biol. Chem. 2005; 280: 32618-32624
        • Apkarian A.V.
        • Bushnell M.C.
        • Treede R.D.
        • Zubieta J.K.
        Eur. J. Pain. 2005; 9: 463-484
        • Oertel B.G.
        • Preibisch C.
        • Wallenhorst T.
        • Hummel T.
        • Geisslinger G.
        • Lanfermann H.
        • Lotsch J.
        Clin. Pharmacol. Ther. 2008; 83: 577-588
        • Price D.D.
        Science. 2000; 288: 1769-1772
        • Coghill R.C.
        • Sang C.N.
        • Maisog J.M.
        • Iadarola M.J.
        J. Neurophysiol. 1999; 82: 1934-1943
        • Bornhovd K.
        • Quante M.
        • Glauche V.
        • Bromm B.
        • Weiller C.
        • Buchel C.
        Brain. 2002; 125: 1326-1336
        • Skarke C.
        • Kirchhof A.
        • Geisslinger G.
        • Lotsch J.
        Clin. Chem. 2004; 50: 640-644
        • Bradford M.M.
        Anal. Biochem. 1976; 72: 248-254
        • Gonzalez-Maeso J.
        • Rodriguez-Puertas R.
        • Gabilondo A.M.
        • Meana J.J.
        Eur. J. Pharmacol. 2000; 390: 25-36
        • Sim L.J.
        • Selley D.E.
        • Xiao R.
        • Childers S.R.
        Eur. J. Pharmacol. 1996; 307: 97-105
        • Maher C.E.
        • Selley D.E.
        • Childers S.R.
        Biochem. Pharmacol. 2000; 59: 1395-1401
        • Ko M.C.
        • Lee H.
        • Harrison C.
        • Clark M.J.
        • Song H.F.
        • Naughton N.N.
        • Woods J.H.
        • Traynor J.R.
        J. Pharmacol. Exp. Ther. 2003; 306: 179-186
        • Neilan C.L.
        • Janvey A.J.
        • Bolan E.
        • Berezowska I.
        • Nguyen T.M.
        • Schiller P.W.
        • Pasternak G.W.
        J. Pharmacol. Exp. Ther. 2003; 306: 430-436
        • Zubieta J.K.
        • Heitzeg M.M.
        • Smith Y.R.
        • Bueller J.A.
        • Xu K.
        • Xu Y.
        • Koeppe R.A.
        • Stohler C.S.
        • Goldman D.
        Science. 2003; 299: 1240-1243
        • Berthele A.
        • Platzer S.
        • Jochim B.
        • Boecker H.
        • Buettner A.
        • Conrad B.
        • Riemenschneider M.
        • Toelle T.R.
        Neuroimage. 2005; 28: 185-193
        • Baumgartner U.
        • Buchholz H.G.
        • Bellosevich A.
        • Magerl W.
        • Siessmeier T.
        • Rolke R.
        • Hohnemann S.
        • Piel M.
        • Rosch F.
        • Wester H.J.
        • Henriksen G.
        • Stoeter P.
        • Bartenstein P.
        • Treede R.D.
        • Schreckenberger M.
        Neuroimage. 2006; 30: 692-699
        • Pilapil C.
        • Welner S.
        • Magnan J.
        • Gauthier S.
        • Quirion R.
        Brain Res. Bull. 1987; 19: 611-615
        • Rodriguez-Puertas R.
        • Gonzalez-Maeso J.
        • Meana J.J.
        • Pazos A.
        Neuroscience. 2000; 96: 169-180
        • Pan Y.X.
        • Xu J.
        • Mahurter L.
        • Xu M.
        • Gilbert A.K.
        • Pasternak G.W.
        Biochem. Biophys. Res. Commun. 2003; 301: 1057-1061
        • Pan L.
        • Xu J.
        • Yu R.
        • Xu M.M.
        • Pan Y.X.
        • Pasternak G.W.
        Neuroscience. 2005; 133: 209-220
        • Chakrabarti S.
        • Prather P.L.
        • Yu L.
        • Law P.Y.
        • Loh H.H.
        J. Neurochem. 1995; 64: 2534-2543
        • Kleuss C.
        • Scherubl H.
        • Hescheler J.
        • Schultz G.
        • Wittig B.
        Nature. 1992; 358: 424-426