Abstract
[Dmt1]DALDA (H-Dmt-d-Arg-Phe-Lys-NH2; Dmt = 2′,6′-dimethyltyrosine) binds with high affinity and selectivity to the μ opioid receptor and is a surprisingly potent and long-acting analgesic, especially after intrathecal administration. In an attempt to better understand the unique pharmacological profile of [Dmt1]DALDA, we have prepared [3H][Dmt1]DALDA and compared its binding properties with that of [3H]DAMGO ([d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin). Kinetic studies revealed rapid association of [3H][Dmt1]DALDA when incubated with mouse brain membranes (K+1 = 0.155 nM–1 min–1). Dissociation of [3H][Dmt1]DALDA was also rapid (K–1 = 0.032 min–1) and indicated binding to a single site. [3H][Dmt1]DALDA binds with very high affinity to human μ opioid receptor (hMOR) (Kd = 0.199 nM), and Kd and Bmax were reduced by sodium but not Gpp(NH)p [guanosine 5′-(β,γ-imido)triphosphate]. Similar Kd values were obtained in brain and spinal cord tissues and SH-SY5Y cells. The hMOR:hDOR (human δ opioid receptor) selectivity of [Dmt1]DALDA (∼10,000) is 8-fold higher than DAMGO. However, [Dmt1]DALDA is less selective than DAMGO against hKOR (human κ opioid receptor) (26-versus 180-fold). The Ki values for a number of opioid ligands were generally higher when determined by competitive displacement binding against [3H][Dmt1]DALDA compared with [3H]DAMGO, with the exception of Dmt1-substituted peptide analogs. All Dmt1 analogs showed much higher affinity for the μ receptor than corresponding Tyr1 analogs. [35S]GTPγS (guanosine 5′-O -(3-[35S]thio)triphosphate) binding showed that [Dmt1]DALDA and DAMGO are full agonists at hMOR and hDOR but are only partial agonists at hKOR. The very high affinity and selectivity of [3H][Dmt1]DALDA for the μ receptor, together with its very low nonspecific binding (10–15%) and metabolic stability, make [3H][Dmt1]DALDA an ideal radioligand for labeling μ receptors.
Opioid receptors belong to the superfamily of guanine nucleotide binding protein-coupled receptors. Early pharmacological and biochemical studies led to the proposal of three major subtypes of opioid receptors (μ, δ, and κ), which were subsequently confirmed by molecular cloning efforts (Snyder and Pasternak, 2003). The availability of the cloned receptors (MOR, DOR, and KOR) allows studies of individual receptor subtypes with regard to receptor signaling pathways and pharmacological profiles. However, ligands with high selectivity for the individual receptor subtypes are necessary for the study of their functional roles in biological tissues or animals and as therapeutic agents for clinical use. In particular, highly selective radioligands are invaluable for quantifying receptor population in biological tissues and for characterization of novel ligands in competitive displacement binding assays.
[Dmt1]DALDA (H-Dmt-d-Arg-Phe-Lys-NH2; where Dmt = 2′,6′-dimethyltyrosine) is a new dermorphin analog that has very high affinity and selectivity for the μ receptor (Schiller et al., 2000). In competitive displacement binding studies against [3H]DAMGO ([d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin) in mouse brain membranes, the Ki for [Dmt1]DALDA was found to be 0.143 ± 0.015 nM at the μ receptor. By comparing its Ki against [3H]DAMGO binding and [3H]DSLET ([d-Ser2,Leu5]-enkephalin-Thr) binding, the μ:δ selectivity of [Dmt1]DALDA was determined to be 14,700. [Dmt1]DALDA also displayed good μ:κ selectivity in displacement binding against [3H]U69,593, with Kiκ/Kiμ = 156. In comparison, the affinity of DAMGO for the μ opioid receptor was reported to be ∼1.2 nM, with μ:δ selectivity of only 1050 (Schiller et al., 2000). [Dmt1]DALDA behaved as a full agonist in the guinea pig ileum assay (Schiller et al., 2000).
[Dmt1]DALDA is a highly potent and long-acting analgesic after both spinal or supraspinal administration, although it is more potent when given intrathecally (Neilan et al., 2001; Shimoyama et al., 2001; Riba et al., 2002; Zhao et al., 2002). The potency of [Dmt1]DALDA in the spinal cord (1000–3000 times that of morphine) is substantially higher than would be suggested by its affinity at the μ receptor (only 7-fold higher than morphine). The extraordinary potency of [Dmt1]DALDA in the spinal cord, and the inability of naloxonazine (μ1 antagonist) to inhibit supraspinal [Dmt1]DALDA, led to the suggestion that [Dmt1]DALDA and morphine may act at different μ receptors (Neilan et al., 2001).
In an attempt to better understand the unique pharmacological profile of [Dmt1]DALDA, we have prepared [3H][Dmt1]DALDA and compared its binding properties with that of [3H]DAMGO. We have also compared the binding characteristics of [Dmt1]DALDA in membrane preparations expressing cloned human opioid receptors (hMOR, hDOR, and hKOR), as well as in membranes prepared from SH-SY5Y cells (human neuroblastoma cell line) and brain and spinal cord tissues from mice and rats. Finally, we have compared the potency and intrinsic activity of [Dmt1]DALDA and DAMGO in activation of G proteins as measured by [35S]GTPγS binding.
Materials and Methods
Drugs and Chemicals. [Dmt1]DALDA was synthesized according to methods described previously (Schiller et al., 1989, 2000). DAMGO, [d-Pen2,d-Pen5]-enkephalin (DPDPE), [3H]DPDPE (42 Ci/mmol, 0.5mCi/ml), [d-Ala2]deltorphin II (H-Tyr-d-Ala-Phe-Glu-Val-Val-Gly-NH2) and [trans-(±)-3,4-dichloro-N-methyl-[2-(1-pyrolidinyl)-cyclohexyl] benzeneacetamide (U50,488H) were supplied by the National Institute on Drug Abuse (Rockville, Maryland). [3H]DAMGO (50 Ci/mmol, 1.0 mCi/ml), (5α,7α,8α)-(–)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl] benzeneacetamide; 59.0 Ci/mmol, 1.0 mCi/ml) ([3H]U69,593) and [35S]GTPγ S (1000–1200 Ci/mmol, 1.0 mCi/ml) was purchased from Amersham Biosciences Inc. (Piscataway, NJ). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).
Preparation of [3H][Dmt1]DALDA. For the preparation of [Dmt1]DALDA in tritiated form, a precursor peptide containing 2′,6′-dimethyl-3′,5′-diiodotyrosine [Tyr(2′,6′-Me2,3′,5′-I2)] needed to be synthesized. Fmoc-Dmt-OH was iodinated by treatment with I2 in the usual manner to yield Fmoc-Tyr(2′,6′-Me2,3′,5′-I2)-OH. This protected amino acid was then used in the solid phase synthesis of H-Tyr(2′,6′-Me2,3′,5′-I2)-d-Arg-Phe-Lys-NH2 according to a protocol published elsewhere (Schiller et al., 2000). The peptide was purified by preparative reversed-phase chromatography and its structure was confirmed by fast atom bombardment mass spectrometry. Catalytic tritiation of this precursor peptide was performed at the Institute of Isotopes (Budapest, Hungary), resulting in a product with a specific radioactivity of 47.18 Ci/mmol.
Tissue Preparation. Male CD-1 mice (25–30 g) and Sprague-Dawley rats (250–300 g) were purchased from Charles River Laboratories, Inc. (Wilmington, MA). All studies were conducted in accordance with guidelines approved by the Institution for the Care and Use of Animals at Weill Medical College of Cornell University. Mice and rats were decapitated, and brains and spinal cords were removed and stored at –80°C until being used. On the day of experiment, tissue was thawed in 30 volumes of ice-cold 50 mM Tris-HCl buffer (0.5 mM EDTA, pH7.4), homogenized for 10 s and centrifuged at 40,000g for 20 min at 4°C. After this process was repeated a second time, the pellet was resuspended in Tris-HCl buffer (pH 7.4) at a final concentration of 1.5 to 2.0 mg protein/ml. Protein concentrations were determined by the Bradford procedure (Bio-Rad, Hercules, CA).
Preparation of Cell Membranes. Membranes prepared from either CHO-K1 cells transfected with hMOR or hDOR, or HEK293 cells transfected with hKOR were purchased from PerkinElmer Life Sciences (Boston, MA). SH-SY5Y cells were cultured in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and harvested after growing for 4 days to 95% confluence, centrifuged at 500g for 5 min, and stored in –80°C. On the day of the experiment, cells were homogenized in Tris-HCl buffer (0.5 mM EDTA, pH 7.4) and centrifuged at 40,000g for 20 min at 4°C. The pellets were resuspended in Tris-HCl buffer (pH 7.4) at a final concentration of 0.6 mg/ml. Protein concentrations were determined by the Bradford procedure (Bio-Rad).
Radioligand Binding Assay. Binding assays were performed in 50 mM Tris-HCl buffer (pH 7.4) in a final volume of 0.6 to 1.2 ml. The amount of protein added varied from 12 to 15 μg for membranes derived from transfected CHO or HEK cells to 100 to 400 μg in the case of brain or spinal cord membranes. Free radioligand was separated from bound radioligand by rapid filtration through GF/B filters preimmersed for 45 min in 0.2% PEI solution (Brandel, Inc., Gaithersburg, MD). Filters were washed three times with 3 ml of Tris-HCl buffer. Radioactivity was determined by liquid scintillation counting. All binding experiments were carried out in triplicate, and the results represent mean ± S.E. from four to six experiments.
Saturation Binding. Aliquots of membrane homogenates were incubated with [3H][Dmt1]DALDA or [3H]DAMGO for 60 min at 25°C and 37°C, respectively. Nonspecific binding was assessed by including 0.1 μM unlabeled [Dmt1]DALDA or 10 μM naloxone. Equilibrium dissociation constant (Kd) and receptor number (Bmax) were determined using a one-site binding equation and nonlinear regression (GraphPad Software Inc., San Diego, CA).
Binding Kinetics. The association rate constant for [3H][Dmt1]DALDA binding was determined by incubating 0.20 nM [3H][Dmt1]DALDA with mouse brain membranes at 25°C for various times up to 60 min. The observed rate constant (Kob) was determined by fitting the specific binding data to the monoexponential equation Y = Ymax (1 – exp(–Kobt)), where t is the time from onset of association and Ymax is the maximum specific binding at this concentration. Dissociation of the binding of [3H][Dmt1]DALDA to mouse brain membranes was achieved by the addition of high concentrations of unlabeled [Dmt1]DALDA after the 60 min incubation, and the reaction mixture stopped at various times by rapid filtration. The dissociation rate constant (K–1) was calculated from exponential decay analysis: Y = Ymax*exp(–K–1t). The association rate constant (K+1) was determined from K+1 = (Kob – K–1)/[L], where [L] is the radioligand concentration.
Competitive Displacement Binding. Binding affinities of other opioid ligands to hMOR was determined by competitive displacement binding with graded concentrations of unlabeled ligand incubated with 0.10 nM [3H][Dmt1]DALDA for 60 min at 25°C. Nonspecific binding was determined using 0.1 μM [Dmt1]DALDA. The selectivity of [Dmt1]DALDA and DAMGO for μ, δ, and κ receptors was determined with the use of competitive displacement binding assays carried out with membranes expressing hMOR, hDOR, or hKOR, respectively. hMOR membranes were incubated with 0.100 nM [3H][Dmt1]DALDA and graded concentrations of unlabeled [Dmt1]DALDA for 60 min at 25°C, and nonspecific binding determined using 0.1 μM [Dmt1]DALDA. hDOR membranes were incubated with 2 nM [3H]DPDPE and graded concentrations of [Dmt1]DALDA for 120 min at 25°C, and nonspecific binding determined using 2 μM unlabeled DPDPE. hKOR membranes were incubated with 0.8 nM [3H]U69,593 and graded concentrations of unlabeled [Dmt1]DALDA for 80 min at 25°C, and nonspecific binding determined with 10 μM naloxone. For all competitive binding assays, IC50 was determined from the displacement curves using a one-site model and nonlinear regression (GraphPad Software Inc.). Ki values were calculated from the obtained IC50 values by means of the Cheng and Prusoff equation, Ki = IC50/(1 + L/Kd), where L and Kd are the concentration and affinity of the radiolabeled ligand in the assay (Cheng and Prusoff, 1973).
[35S]GTPγS Binding Assay. The potency and intrinsic activity of [Dmt1]DALDA and DAMGO at hMOR, hDOR, and hKOR were determined using [35S]GTPγS binding. Aliquots of membrane homogenates (6–10 μg of protein) were incubated with 50 pM [35S]GTPγS and 30 μM GDP in 1 ml Tris buffer (50 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 0.1% bovine serum albumin, pH 7.4) in the presence of varying concentrations of [Dmt1]DALDA or DAMGO for 60 min at 30°C. For DOR and KOR membranes, the concentration of GDP was 60 and 10 μM, respectively. Nonspecific binding was determined using 10 μM unlabeled GTPγS. Free radioligand was separated from bound radioligand by rapid filtration as described above. Potency (EC50) and intrinsic activity (Emax) were determined from dose-response curves analyzed using nonlinear regression (GraphPad Software Inc.). Full agonist activity at the three receptors were determined using DAMGO for hMOR, deltorphin II, at hDOR, and U50,488H at hKOR.
Results
Saturation Binding of [3H][Dmt1]DALDA. Saturation binding of [3H][Dmt1]DALDA was carried out using membranes prepared from cells transfected with hMOR (CHO-K1/hMOR). Specific binding of [3H][Dmt1]DALDA was saturable and of high affinity (Fig. 1). The extent of nonspecific binding was similar whether it was determined using excess unlabeled [Dmt1]DALDA or naloxone, and was only 10 to 15% of total binding at radioligand concentrations near Kd value. The Kd and Bmax were determined to be 0.199 ± 0.019 nM, and 926 ± 55 fmol/mg protein, respectively (n = 6).
Saturation binding with [3H][Dmt1]DALDA was also carried out using membranes prepared from SH-SY5Y cells, rat brain, and spinal cord, and mouse brain and spinal cord (Fig. 1). A one-site binding model provided good fit for all binding curves. The Kd and Bmax values for these various membranes are summarized in Table 1. The Kd for [3H][Dmt1]DALDA was comparable in all membrane preparations and was ∼0.10 to 0.20 nM. Receptor density was 6- to 9-fold lower in brain and spinal cord compared with the receptor number expressed on CHO cells. The number of binding sites was very low in undifferentiated SH-SY5Y cells.
Comparison of [3H][Dmt1]DALDA and [3H]DAMGO Binding. The binding of [3H][Dmt1]DALDA was compared with the binding of [3H]DAMGO by using membranes prepared from hMOR and mouse brain (Fig. 2). Nonspecific binding of [3H]DAMGO (∼10%) was similar to that of [3H][Dmt1]DALDA. The affinity of [3H]DAMGO (Kd = 3.10 ± 0.53 nM) for hMOR was 26-fold lower compared with [3H][Dmt1]DALDA, whereas Bmax was a little lower at 799 ± 47 fmol/mg protein (n = 5). With mouse brain membranes, the Kd for [3H][Dmt1]DALDA and [3H]DAMGO were estimated to be 0.123 ± 0.012 nM (n = 5) and 1.59 ± 0.14 nM (n = 4), respectively. The receptor density (Bmax) determined with [3H][Dmt1]DALDA (130 ± 9 fmol/mg protein) was not different from that determined using [3H]DAMGO (115 ± 3 fmol/mg protein).
Effects of Sodium and Gpp(NH)p on [3H][Dmt1]DALDA Binding. Both Kd and Bmax of [3H][Dmt1]DALDA binding to mouse brain membranes were significantly affected by the addition of NaCl (100 mM) and Gpp(NH)p (10 μM) (Table 2). However, addition of Gpp(NH)p alone, even at 100 μM, had no effect on either Kd or Bmax, whereas the addition of NaCl alone resulted in almost 50% decrease in Bmax and 2- to 3-fold increase in Kd. When the binding assay was carried out under conditions used for [35S]GTPγS binding (100 mM NaCl and 30 μM GDP), Kd and Bmax were 0.926 ± 0.17 nM and 940 ± 66 fmol/mg protein, respectively (n = 6).
Kinetics of [3H][Dmt1]DALDA Binding. Kinetic studies revealed rapid association and dissociation of [3H][Dmt1]DALDA when incubated with mouse brain membrane homogenates. The rate of binding of [3H][Dmt1]DALDA was concentration-dependent, and specific binding of [3H][Dmt1]DALDA reached steady state after 50 min of incubation at concentration of 0.20 nM (Fig. 3A). Dissociation of specifically bound radioligand was initiated by the addition of 10 μM unlabeled [Dmt1]DALDA. Nonlinear regression of the dissociation curve showed that it was best fit with a monoexponential equation and the dissociation half-life was estimated to be ∼21 min). The association rate constant (K+1) and dissociation rate constant (K–1) were determined to be 0.1551 ± 0.0402 nM–1 min–1 (n = 6) and 0.0320 ± 0.0074 min–1, respectively (n = 6). The Kd value was calculated from these values to be 0.206 nM.
Competitive Displacement Binding. [3H][Dmt1] DALDA binding to mouse brain membranes was completely displaced by increasing concentrations of the opioid ligands shown in Table 3. The Ki values, and the corresponding Ki values determined by displacement of [3H]DAMGO binding are also summarized in Table 3 (Schiller et al., 2000). In general, there was excellent correlation between Ki ([3H][Dmt1]DALDA) and Ki ([3H]DAMGO) for those peptide analogs with [Dmt1]. However, Ki([3H][Dmt1]DALDA) were higher than Ki([3H]DAMGO) for all other ligands. This discrepancy resulted in significantly different relative affinities when the peptide ligands were compared with morphine. The relative affinity of [Dmt1]DALDA was 7 times greater than morphine when determined using Ki([3H]DAMGO) but 35-fold when determined using Ki([3H][Dmt1]DALDA). Substitution of Tyr1 with Dmt1 increased affinity of these dermorphin (1–4)-tetrapeptide analogs 10–20-fold, with all [Dmt1] analogs having very high affinity binding (Ki ∼0.15 nM).
Binding of [Dmt1]DALDA and DAMGO to MOR, DOR, and KOR Membranes. The selectivity of [Dmt1]DALDA and DAMGO for μ, δ, and κ receptors were determined using hMOR, hDOR, or hKOR membranes. Figure 4A illustrates the competitive displacement curves for [Dmt1]DALDA against [3H][Dmt1]DALDA, [3H]DPDPE, and [3H]U69,593 binding to hMOR, hDOR, and hKOR membranes, respectively. Figure 4B shows the corresponding competitive displacement curves for DAMGO. The Kd for [3H][Dmt1]DALDA, [3H]DPDPE, and [3H]U69,593 in hMOR, hDOR, and hKOR were 0.199 ± 0.019 nM (n = 6), 1.95 ± 0.20 nM (n = 4), and 0.694 ± 0.08 nM (n = 3), respectively. The Ki value for [Dmt1]DALDA and DAMGO at the three receptors are summarized in Table 4. The μ:δ selectivity was almost 10-fold higher for [Dmt1]DALDA than DAMGO, whereas μ:κ selectivity was higher for DAMGO.
Effects of [Dmt1]DALDA in Stimulation of GTPγS Binding. The potency and intrinsic activity of [Dmt1]DALDA and DAMGO at hMOR, hDOR, and hKOR were assessed by their ability to stimulate [35S]GTPγS binding and compared with full agonists (DAMGO for hMOR, deltorphin II for hDOR, and U50,488H for hKOR). Figure 5 illustrates the dose-response curves for [Dmt1]DALDA and DAMGO in hMOR, hDOR, and hKOR membranes. [Dmt1]DALDA and DAMGO acted as agonist at all three receptors. The results are summarized in Table 5.
The EC50 value for [Dmt1]DALDA at hMOR was 12-fold lower than that of DAMGO. The Emax for [Dmt1]DALDA (335.3%) was not significantly different from the maximal effect elicited by DAMGO (362.3%). The maximal effect elicited by [Dmt1]DALDA (158.0 ± 7.5%) was also comparable with that elicited by DAMGO (151.0 ± 5.3%) when [35S]GTPγS binding was carried out using mouse brain membranes. At hDOR, both [Dmt1]DALDA and DAMGO were able to elicit a maximal response that was larger than that elicited by deltorphin II. By comparing the potency of [Dmt1]DALDA and DAMGO at hMOR and hDOR, their MOR:DOR selectivity was determined to be 118 and 7, respectively. In contrast, both [Dmt1]DALDA and DAMGO were only partial agonists at KOR, with Emax being 56.1 and 79.3%, respectively, compared with U50,488H. The MOR: KOR selectivity was 29 for [Dmt1]DALDA and 37 for DAMGO.
Discussion
The affinity of [Dmt1]DALDA for μ, δ, and κ receptors was originally ascertained by displacement of [3H]DAMGO and [3H]DSLET binding from rat brain membranes, and displacement of [3H]U69,593 binding from guinea pig brain membranes, respectively (Schiller et al., 2000). However, brain tissues express multiple subtypes of opioid receptors and these radioligands lack specificity. The availability of [3H][Dmt1]DALDA and cells transfected with hMOR, hDOR, or hKOR allowed us to fully characterize the binding and G protein activation of [Dmt1]DALDA at μ, δ,and κ receptors.
Kinetic studies revealed rapid association of [3H][Dmt1]DALDA when incubated with mouse brain membranes, with K+1 of 0.155 nM–1 min–1. This is a bit faster than the association constant reported for [3H]diprenorphine (0.116 nM–1 min–1) (Ott et al., 1986), [3H]H-Tyr-d-Ala-Phe-Phe-NH2 (0.117 nM–1 min–1) (Spetea et al., 1998), and [3H]DAMGO (0.0846 nM–1 min–1) (Zajac and Roques, 1985). Dissociation of [3H][Dmt1]DALDA was also rapid and binding declined in a monoexponential manner (K–1 = 0.0320 min–1; t1/2 = 21.7 min), suggesting that [3H][Dmt1]DALDA was binding to a single site. This is in contrast to previous reports suggesting two different sites for [3H]diprenorphine (Ott et al., 1986) and [3H]DAMGO (Brown and Pasternak, 1998).
Equilibrium binding studies showed that [3H][Dmt1]DALDA binds with very high affinity to hMOR with Kd of 0.199 nM, and this is in agreement with the Kd calculated from the kinetic studies using mouse brain membranes (Kd = K–1/K+1 = 0.206 nM), as well as the Ki determined against [3H]DAMGO binding (0.143 nM) (Schiller et al., 2000). Receptor numbers are very high in these membranes, and the Bmax determined with [3H][Dmt1]DALDA (926 fmol/mg protein) was similar to the Bmax obtained with [3H]diprenorphine (1080 fmol/mg protein) in these membranes (provided by the manufacturer). The relative affinity of [3H][Dmt1]DALDA for hMOR and hDOR (∼10,000) is similar to the earlier value of 14,700 determined by displacement binding with mouse brain membranes (Schiller et al., 2000), making [Dmt1]DALDA 8-fold more selective than DAMGO. However, [3H][Dmt1]DALDA also binds hKOR with nanomolar affinity, thus resulting in a μ:κ selectivity (26) that is lower than previously determined by displacement binding (156) (Schiller et al., 2000), and is lower compared with DAMGO (180).
The high affinity and selectivity of [3H][Dmt1]DALDA for the μ receptor, together with its low nonspecific binding, makes it an ideal radioligand for labeling μ receptors in biological tissues. The Kd for [3H][Dmt1]DALDA obtained from equilibrium binding studies using brain and spinal cord tissues from mice and rats were the same as that determined with hMOR. The low nonspecific binding of [3H][Dmt1]DALDA made it particularly good for quantifying low receptor numbers, such as that expressed on SH-SY5Y cells.
We have compared the use of [3H][Dmt1]DALDA with [3H]DAMGO for evaluating the affinity of other opioid ligands in binding to mouse brain membranes. The Ki determined against [3H][Dmt1]DALDA were generally higher than the Ki determined against [3H]DAMGO binding, with the exception of peptide analogs that had a Dmt1 substitution for Tyr1. These data suggest that the Dmt1 analogs may bind to a different site and the other ligands do not express the same affinity for this site. All of the Dmt1 peptide analogs showed much higher affinity for the μ receptor compared with the corresponding Tyr1 analogs. The substitution of Dmt1 for Tyr1 has been shown to consistently increase the binding affinity toward both μ and δ receptors, but the mechanism remains uncertain (Hansen, Jr. et al., 1992; Sasaki et al., 1999; Schiller et al., 2000; Harrison et al., 2003).
Sodium and guanine nucleotides are known to diminish the binding of opioid agonists but not antagonists (Pert et al., 1973). Sodium has been reported to decrease the binding affinity of [3H]DAMGO, and either increase or had no change on Bmax (Werling et al., 1986; Bolger et al., 1987; Krumins et al., 1993; Brown and Pasternak, 1998). The presence of Gpp(NH)p also decreased binding affinity of [3H]DAMGO but had little effect on Bmax (Werling et al., 1986; Bolger et al., 1987; Krumins et al., 1993; Brown and Pasternak, 1998). As expected, Kd was increased 3-fold when [3H][Dmt1]DALDA binding was carried out in the presence of sodium and guanine nucleotides. Surprisingly, the reduced affinity in [3H][Dmt1]DALDA binding was entirely due to the presence of sodium, whereas Gpp(NH)p itself had no effect. Although guanine nucleotides inhibit the binding of most opioid agonists, the binding of etorphine was also not affected by GTP (Childers and Snyder, 1980). The lack of effect of Gpp(NH)p suggests that [3H][Dmt1]DALDA binds to both the G protein-coupled and uncoupled receptor with similar affinity. This may account for the very high in vivo analgesic potency of [Dmt1]DALDA compared with morphine.
Our results also show that [Dmt1]DALDA is an agonist at all three receptor subtypes. [35S]GTPγS binding revealed that [Dmt1]DALDA is a full agonist at hMOR and hDOR, although its potency is 118-fold higher at hMOR. In contrast, DAMGO only showed 7-fold selectivity for hMOR versus hDOR. It is interesting that [Dmt1]DALDA showed significantly higher MOR:DOR selectivity in receptor binding assays compared with GTPγS binding assays (9707 versus 118). This discrepancy can not be explained by the different conditions used for the two assays because the Kd of [3H][Dmt1]DALDA was only increased slightly in the presence of 100 mM NaCl and 30 μM GDP. It is possible, however, that the coupling efficiency of DOR is much higher than MOR. This possibility is supported by the fact that the MOR: DOR selectivity of DAMGO was also much greater in the radioligand binding assay compared with [35S]GTPγS binding (1212 versus 7). Both [Dmt1]DALDA and DAMGO were partial agonists at hKOR, but the Emax for [Dmt1]DALDA (56%) was lower compared with DAMGO (79%). Thus, the efficacy of [Dmt1]DALDA at the κ receptor is limited despite its high binding affinity.
In summary, [3H][Dmt1]DALDA seems to be a superior radioligand for labeling μ receptors in biological tissues. Its advantages include high affinity and high selectivity, low nonspecific binding, and metabolic stability (Szeto et al., 2001). In addition, [Dmt1]DALDA is a potent and long-acting analgesic after intrathecal and subcutaneous administration (Neilan et al., 2001; Shimoyama et al., 2001; Zhao et al., 2002; Riba et al., 2002), and can protect the heart against ischemia-reperfusion injury (Wu et al., 2002).
Footnotes
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This work was supported, in part, by a multicenter program project grant (DA08924) from the National Institute on Drug Abuse.
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ABBREVIATIONS: hMOR, cloned human μ opioid receptor; hDOR, cloned human δ opioid receptor; hKOR, cloned human κ opioid receptor; [Dmt1]DALDA, H-Dmt-d-Arg-Phe-Lys-NH2; Dmt = 2′,6′-dimethyltyrosine; DAMGO, [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin; DSLET, [d-Ser2,Leu5]-enkephalin-Thr; Fmoc, N-(9-fluorenyl)methoxycarbonyl; U69,593, (5α,7α,8β)-(+)-N-methyl-N-(7-[1-pyrrolidinyl]-1-oxaspiro[4.5]dec-8-yl)-benzeneacetamide; GTPγS, guanosine 5′-O-(3-thio)triphosphate; DPDPE, [d-Pen2,d-Pen5]-enkephalin; U50,488H, [trans-(±)-3,4-dichloro-N-methyl-[2-(1-pyrolidinyl)-cyclohexyl] benzeneacetamide; HEK, human embryonic kidney; CHO, Chinese hamster ovary; Gpp(NH)p, guanosine 5′-(β,γ-imido)triphosphate.
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DOI: 10.1124/jpet.103.054775.
- Received May 21, 2003.
- Accepted August 22, 2003.
- The American Society for Pharmacology and Experimental Therapeutics