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NEUROPHARMACOLOGY

Studies of µ-, -, and -Opioid Receptor Density and G Protein Activation in the Cortex and Thalamus of Monkeys

Departments of Anesthesiology (N.N.N.) and Pharmacology (M.C.H.K., H.L., C.H., M.J.C., H.F.S., J.H.W., J.R.T.), The University of Michigan Medical School, Ann Arbor, Michigan

Received February 18, 2003; accepted April 1, 2003.

	Abstract

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The aim of this study was to investigate the relative density of µ-, -, and -opioid receptors (MOR, KOR, and DOR) and guanosine 5'-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPS) binding stimulated by full agonists in cortical and thalamic membranes of monkeys. The binding parameters [B_max (femtomoles per milligram)/K_d (nanomolar)] were as follows: [³H][D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (DAMGO) (MOR; 80/0.7), [³H]U69593 [(5,7,8)-(–)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl) benzeneacetamide] (KOR; 116/1.3), and [³H][D-Pen²,D-Pen⁵]-enkephalin (DPDPE) (DOR; 87/1.3) in the cortex; [³H]DAMGO (147/0.9), [³H]U69593 (75/2.5), and [³H]DPDPE (22/2.0) in the thalamus. The relative proportions of MOR, KOR, and DOR in the cortex were 28, 41, and 31% and in the thalamus were 60, 31, and 9%. Full selective opioid agonists, DAMGO (EC₅₀ = 532–565 nM) and U69593 [GenBank] (EC₅₀ = 80–109 nM) stimulated [³⁵S]GTPS binding in membranes of cortex and thalamus, whereas SNC80 [(+)-4-[(R)--((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethyl-benzamide] (DOR; EC₅₀ = 68 nM) was only active in cortical membranes. The magnitudes of [³⁵S]GTPS binding stimulated by these agonists were similar in the cortex, ranging from 17 to 25% over basal binding. In the thalamus, DAMGO and U69593 [GenBank] increased [³⁵S]GTPS binding by 44 and 23% over basal, respectively. Opioid agonist-stimulated [³⁵S]GTPS binding was blocked selectively by antagonists for MOR, KOR, and DOR. The amount of G protein activated by agonists was highly proportional to the relative receptor densities in both regions. These results distinguish the ability of opioid agonists to activate G proteins and provide a functional correlate of ligand-binding experiments in the monkey brain. In particular, the relative densities of opioid receptor binding sites in the two brain areas reflect their functional roles in the pharmacological actions of opioids in the central nervous system of primates.

Several investigations in the monkey have shown that systemic administration of full MOR or KOR agonists produces profound antinociception against acute noxious stimuli, but that high-efficacy DOR agonists have relatively weak antinociceptive effects (Negus et al., 1994; Ko et al., 1998; Allen et al., 2002). Given that MOR, KOR, and DOR agonists all have inhibitory functions on nociceptive pathways, it is unknown what factors may contribute to this differential antinociceptive effectiveness among these full opioid agonists. In particular, it is pivotal to investigate whether there is a difference in the density of opioid receptor types in the neural substrates relevant to nociceptive pathways and in their functional activity at the level of the receptor-G protein interaction.

The opioid agonist-mediated stimulation of [³⁵S]GTPS binding in cell lines expressing specific receptors has provided a functional measurement of agonist occupation of MOR, KOR, and DOR and offered a simple method for the determination of efficacy of opioid agonists (Traynor and Nahorski, 1995; Clark et al., 1997; Selley et al., 1998; Remmers et al., 1999). However, it is not yet clear whether full opioid agonists would manifest the same or different pharmacological profiles in primate brain regions containing a mixture of different densities of MOR, KOR, and DOR. For example, differences could be attributed to the presence of receptor subtypes (Ko et al., 1998) and/or homo- or heterodimers (Jordan and Devi, 1999). Studies characterizing opioid agonist-stimulated [³⁵S]GTPS binding in monkey brain membranes are particularly valuable because they will provide a pharmacological explanation for the central actions of both exogenous and endogenous opioid ligands in primates.

Imaging studies in humans show that both cortex and thalamus are significantly involved in nociceptive pathways (Davis et al., 1998; Casey et al., 2000; Peyron et al., 2000; Zubieta et al., 2001). In particular, the thalamus is the main neural substrate in nociceptive pathways and plays an integral role for both spinothalamic and thalamocortical pathways (Bushnell et al., 1993; Craig et al., 1994; Shi and Apkarian, 1995; Rausell et al., 1998). The aim of this present study was to measure the density of MOR, KOR, and DOR in the monkey cortex and thalamus. In addition, full opioid agonists selective for each opioid receptor type were used to investigate agonist-stimulated [³⁵S]GTPS binding to observe functional activity at the level of the receptor-G protein in both cortical and thalamic membranes.

	Materials and Methods

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Materials. [³H]DAMGO (50 Ci/mmol), [³H]U69593 (41.4 Ci/mmol), [³H]DPDPE (45 Ci/mmol), [³H]diprenorphine (50 Ci/mmol), and [³⁵S]GTPS (1250 Ci/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA). SNC80 and naltrindole were provided by Dr. K. C. Rice (National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD). DAMGO and naloxone were obtained from National Institute on Drug Abuse (National Institutes of Health). U69593 [GenBank] , nor-binaltorphimine (nor-BNI), GTPS, GDP, and all other biochemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Membrane Preparation. Membranes were isolated from the brains of five adult rhesus monkeys (Macaca mulatta) that weighed between 7.4 and 14.1 kg. The ages of monkeys were between 8 and 13 years except for one monkey that was 22 years old. None of the monkeys were involved in chronic administration of opioids and they had not received any opioid compounds for approximately 4 to 6 months before the current experiments. Monkeys were maintained in accordance with the University Committee on the Use and Care of Animals in the University of Michigan and the Guide for the Care and Use of Laboratory Animals (7th edition) by the Institute of Laboratory Animal Resources (National Academy Press, Washington, DC, revised 1996).

After euthanasia by i.v. pentobarbital (100 mg/kg), each monkey brain was rapidly excised and placed with dry ice before storage at –80°C. The subsequent handling of the tissues was carried out at 4°C. All membranes and vessels were removed from the dorsal surface. The brain tissues were dissected meticulously, washed in 50 mM Tris-HCl buffer, pH 7.4, and then disrupted for 1 min in the ice-cold buffer with a Polytron homogenizer set at power 6.5 (model PT-10; Brinkmann Instruments, Westbury, NY). The homogenized membranes were centrifuged at 18,000g for 15 min. The resulting membrane pellets were resuspended and incubated at 37°C for 40 min to remove endogenous opioids (Wood et al., 1989). The preparation was centrifuged again and the pellet was resuspended in 50 mM Tris-HCl buffer. Aliquots of this suspension, sufficient for experiments on one given day, were frozen at –80°C. Before use, the frozen suspension was quickly thawed and kept on ice. The protein concentration of both cortical and thalamic membrane suspensions was approximately 5 to 9 mg/ml, determined by the method of Bradford (1976) with bovine serum albumin as the standard. Membranes were dissolved with 1 N NaOH for 30 min at room temperature and neutralized with 1 M acetic acid before the protein determination.

Receptor Binding Assay. Saturation binding experiments were performed using [³H]DAMGO, [³H]U69593, [³H]DPDPE, or [³H]diprenorphine (a nonselective opioid ligand), as described previously (Fischel and Medzihradsky, 1981; Emmerson et al., 1994). The assay medium contained membrane protein (250 µg/tube) diluted in Tris-HCl buffer (50 mM Tris-HCl buffer, pH 7.4), 50 µl of Tris-HCl buffer or 50 µM naloxone, and 50 µl of increasing concentrations of radioligand in a final volume of 500 µl. Specific binding of the radioligand was obtained as the difference between binding in the absence and presence of 50 µM naloxone. After incubation for 90 min at 25°C to reach equilibrium, samples were quickly filtered and washed three times with 2 ml of ice-cold 50 mM Tris-HCl buffer, pH 7.4, through 0.1% polyethylenimine-treated glass fiber filters (no. 32; Schleicher & Schuell, Keene, NH) mounted in a cell harvester (Brandel, Inc., Gaithersburg, MD). Filters were placed in 5-ml polypropylene scintillation vials containing 4 ml of Econo-Safe scintillation cocktail for liquid scintillation counting. Experiments were performed two to three times in cortical membranes and once in thalamic membranes from each monkey due to limited availability of tissue. Each experiment was carried out in duplicate.

[³⁵S]GTPS Binding Assay. Agonist stimulation of [³⁵S]GTPS binding was measured as described in Traynor and Nahorski (1995). Membranes (20–60 µg of protein/tube) were incubated in GTPS binding buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, and 10 mM MgCl_2·6H₂O) containing [³⁵S]GTPS (0.1 nM), GDP (100 µM), and varying concentrations (1–30,000 nM) of opioids, either DAMGO, U69593 [GenBank] , or SNC80, in a total volume of 500 µl, for 60 min at 25°C. SNC80 was used for these studies rather than DPDPE because it is more efficacious at stimulating [³⁵S]GTPS binding (Clark et al., 1997). Inhibition of agonist-stimulated [³⁵S]GTPS binding by naloxone (20 nM), nor-binaltorphimine (3 nM), and naltrindole (3 nM) was evaluated by adding antagonist to the membrane 15 min before the agonist. The reaction was terminated by rapidly filtering and washing three times with 2 ml of ice-cold GTPS binding buffer. Filters were placed in scintillation vials containing 4 ml of Econo-Safe scintillation cocktail for liquid scintillation counting. Basal binding was determined from tubes that contained the same volume of GTPS binding buffer without agonist and antagonist. Nonspecific binding was defined as binding of the [³⁵S]GTPS in the presence of 10 µM unlabeled GTPS. Because nonspecific binding was less than 5% of basal binding in these conditions, basal cpm was subtracted from each data point and converted to femtomoles per milligram of protein and the percentage over basal to determine agonist-stimulated [³⁵S]GTPS binding. Experiments were performed at least twice in both cortical and thalamic membranes from each monkey. Each experiment was carried out in duplicate.

Data Analysis. Saturation binding data of [³H]DAMGO, [³H]U69593, [³H]DPDPE, and [³H]diprenorphine were fit to a one-site binding hyperbola using GraphPad Prism (GraphPad Software, Inc., San Diego, CA) to determine K_d and B_max values. [³⁵S]GTPS binding data from two experiments were combined and fit to a sigmoidal curve with a variable slope using GraphPad Prism to determine the EC₅₀ value and maximum stimulation (femtomoles per milligram of protein). Mean values (mean ± S.E.M.) were calculated from individual values (i.e., B_max and the degree of [³⁵S]GTPS stimulation). Data were analyzed by a two-way analysis of variance followed by the Newman-Keuls test for multiple (post hoc) comparisons (p < 0.05 for significance). K_e values for antagonist inhibition were calculated by the following equation: K_e = [nanomolar antagonist]/(dose ratio – 1) where dose ratio is the ratio of the EC₅₀ for an agonist in the presence and absence of the antagonist. In addition, linear regression was made to determine relationship between B_max and [³⁵S]GTPS binding data by pooling the B_max values of radioligands and the magnitudes of [³⁵S]GTPS binding stimulated by corresponding agonists.

	Results

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Receptor Binding Assay. Saturation binding of selective radioligands to monkey brain membranes was studied over an extended range of concentrations. Figure 1 illustrates the apparent affinity and B_max data in cortical membranes from a single monkey, determined using [³H]DAMGO, [³H]U69593, [³H]DPDPE, or [³H]diprenorphine. Nonspecific binding was low and amounted to approximately 20 to 35% of total binding at a nearly saturating concentration of each radioligand. Figure 2 illustrates the apparent affinity and B_max data in the thalamic membranes of a single monkey determined with the same radioligands. Due to small differences in the profile of binding parameters across brain membranes from individual monkeys, the mean data were obtained from individual values and are summarized in Table 1. ANOVA indicates that the differences of B_max values depend on both regions [F(1,4) = 25; p < 0.05] and receptor types [F(2,8) = 102; p < 0.05]. Post hoc comparisons reveal that the mean B_max value of MOR in the cortex is significantly lower than in the thalamus. Mean B_max values of both KOR and DOR in the cortex are significantly higher than in the thalamus. Furthermore, the B_max value of KOR is significantly higher than both of MOR and DOR; and there is no difference between MOR and DOR in the cortex. In contrast, the rank order of B_max values of opioid receptor types is significantly different (i.e., MOR > KOR > DOR) in the thalamus. The MOR, KOR, and DOR were approximately 28, 41, and 31% of the total binding sites in the cortex, respectively. In contrast, the MOR, KOR, and DOR were approximately 60, 31, and 9% of the total binding sites in the thalamus.

View larger version (27K): [in this window] [in a new window]   Fig. 1. Saturation binding of [3H]DAMGO, [3H]U69593, [3H]DPDPE, and [3H]diprenorphine to cortical membranes of one monkey. Specific binding at each concentration of radioligand is shown in the panel, and the Scatchard plot of these data is shown in the corresponding inset. Data from one of three independent experiments are shown. See Materials and Methods and Table 1 for other details.

View larger version (27K): [in this window] [in a new window]   Fig. 2. Saturation binding of [3H]DAMGO, [3H]U69593, [3H]DPDPE, and [3H]diprenorphine to thalamic membranes of one monkey. Specific binding at each concentration of radioligand is shown in the panel, and the Scatchard plot of these data is shown in the corresponding inset. Data from one of three independent experiment are shown. See Materials and Methods and Table 1 for other details.

There was a significant difference in B_max values with [³H]DAMGO and [³H]diprenorphine in the cortex between male and female monkeys. The B_max values obtained using [³H]DAMGO in cortical membranes of male monkeys were significantly higher than those in female monkeys [*p < 0.05; males (n = 3): 89 ± 4.8 versus females (n = 2): 67 ± 2.1 fmol/mg of protein]. Similarly, the B_max values for [³H]diprenorphine in cortical membranes of male monkeys were significantly higher than those in female monkeys (*p < 0.05, males: 345 ± 9 versus females: 280 ± 1 fmol/mg of protein).

[³⁵S]GTPS Binding Assay. Optimal stimulation of the binding of [³⁵S]GTPS (0.1 nM) to monkey brain membranes by opioid agonists was observed at a concentration of 100 µM GDP (data not shown). The average values (± S.E.M.) of basal binding under these conditions were 83.8 ± 7.2 and 71.7 ± 3.8 fmol/mg of protein in cortical and thalamic membranes, respectively. Figure 3 illustrates the stimulation of [³⁵S]GTPS binding by opioid agonists in the cortical membrane of a single monkey. A selective MOR agonist, DAMGO, concentration dependently increased the binding of [³⁵S]GTPS by 19% over basal binding (EC₅₀ = 477 nM). Addition of a low concentration (20 nM) of naloxone (NLX) selective for MOR antagonism produced a 12.1-fold rightward shift in the DAMGO concentration-response curve (EC₅₀ = 5766 nM; Fig. 3, top; Table 2). A selective KOR agonist, U69593 [GenBank] , concentration dependently increased the binding of [³⁵S]GTPS by 20% over basal binding (EC₅₀ = 77 nM). Addition of a selective KOR antagonist, nor-BNI (3 nM), produced an 11.1-fold rightward shift in the U69593 [GenBank] concentration-response curve (EC₅₀ = 858 nM; Fig. 3, middle). In addition, a selective DOR agonist, SNC80, concentration dependently increased the binding of [³⁵S]GTPS by 17% over basal (EC₅₀ = 49 nM). Addition of a selective DOR antagonist, naltrindole (NTI; 3 nM), produced a 24.5-fold rightward shift in the SNC80 concentration-response curve (EC₅₀ = 1190 nM; Fig. 3, bottom). The concentration of each antagonist was selective for its corresponding receptor and did not produce a significant rightward shift of concentration-response curves of other agonists (data not shown). It is worth noting that the degree of DAMGO-stimulated [³⁵S]GTPS binding in the cortex of male monkeys was significantly higher than in the cortex of female monkeys (*p < 0.05, males: 20 ± 1 versus females: 12 ± 2% over basal).

View larger version (20K): [in this window] [in a new window]   Fig. 3. Stimulation of [35S]GTPS binding by opioid agonists in cortical membranes of one monkey. Concentration-response curves of agonist were determined in the absence () or presence () of antagonist, as described in Materials and Methods. EC50 values of DAMGO, U69593 [GenBank] , and SNC80 are 477, 77, and 49 nM, respectively. In the presence of corresponding antagonists, EC50 values of DAMGO, U69593 [GenBank] , and SNC80 are 5766, 858, and 1190 nM, respectively. Shown are mean values ± S.E.M. from two independent experiments, each carried out in duplicate. See Table 2 for other details.

View this table: [in this window] [in a new window]   TABLE 2 Mean values of agonist-stimulated [35S]GTPS binding in both cortical and thalamic membranes of monkeys

View larger version (21K): [in this window] [in a new window]   Fig. 4. Stimulation of [35S]GTPS binding by opioid agonists in thalamic membranes of one monkey. Concentration-response curves of agonist were determined in the absence () or presence () of antagonist, as described in Materials and Methods. EC50 values of DAMGO and U69593 [GenBank] are 433 and 99 nM, respectively. In the presence of corresponding antagonists, EC50 values of DAMGO and U69593 [GenBank] are 6186 and 1135 nM, respectively. EC50 values of SNC80 could not be determined under these conditions. Shown are mean values ± S.E.M. from two independent experiments, each carried out in duplicate. See Table 2 for other details.

There was a linear correlation between the density of opioid receptor binding sites and the degree of opioid agonist-stimulated [³⁵S]GTPS binding in monkey brain membranes. The degree of stimulation of [³⁵S]GTPS binding, presented as the percentage over basal binding, is highly correlated with the density of opioid receptor binding sites in both cortical and thalamic membranes (Fig. 5; r² = 0.83).

View larger version (20K): [in this window] [in a new window]   Fig. 5. Correlation of the density (Bmax) of opioid receptor binding sites and stimulation of [35S]GTPS binding by opioid receptor agonists in monkey brain membranes. Analysis of linear regression reveals r2 = 0.83. The Bmax values were determined by [3H]DAMGO (MOR), [3H]U69593 (KOR), and [3H]DPDPE (DOR). Selective opioid receptor agonists, DAMGO (MOR), U69593 [GenBank] (KOR), and SNC80 (DOR), were used to determine the stimulation of [35S]GTPS binding in membranes, in which data were expressed as percentage of [35S]GTPS stimulation over basal binding. Each symbol represents the value in the corresponding membranes of each monkey.

	Discussion

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The results of this study indicate that the profile of receptor densities of MOR, KOR, and DOR depends on the region in the monkey CNS. The density of MOR in the thalamus is higher than in the cortex. In contrast, the densities of both KOR and DOR in the thalamus are lower than in the cortex. The selectivity of opioid receptor agonists is retained in monkey brain membranes. However, the maximum effect of the full opioid agonists in G protein activation depends on the densities of opioid receptor types in specific brain regions.

In the rhesus monkey brain, the relative proportions of the opioid receptors in the cortex are approximately 28, 41, and 31% for MOR, KOR, and DOR binding sites, respectively. In contrast, MOR, KOR, and DOR are approximately 60, 31, and 9% of the total binding sites in the thalamus. This profile of relative ratios of opioid receptor types in monkey brain is similar to the pattern of receptor densities observed in the cortex and thalamus of humans (Kuhar et al., 1973; Pfeiffer et al., 1982; Mansour et al., 1988). An in situ hybridization study in humans also indicates that probes for MOR, KOR, and DOR generate detectable signals in the cortical regions. However, intense MOR and KOR messenger RNA expression, but no DOR messenger RNA, were detected in the thalamus (Peckys and Landwehrmeyer, 1999). Very low to undetectable levels of DOR messenger RNA expression correspond to low levels of DOR binding sites in several subcortical regions of human brain (Pfeiffer et al., 1982; Peckys and Landwehrmeyer, 1999). Together, these studies indicate that MOR is the predominant type of opioid receptor present in the thalamus of primate species.

We found no evidence for the existence of additional opioid receptor types. The total number of binding sites for the MOR, KOR, and DOR specific agonists represent approximately 89 and 100% of the binding sites labeled by the nonselective antagonist [³H]diprenorphine in cortical and thalamic membranes of the monkey brain, respectively. This finding also confirms that under the conditions of the saturation binding assay the selective ³H-agonists were labeling all opioid receptors. To date, there is no evidence for the existence of additional opioid receptor genes or for splice variants in humans with pharmacological properties characteristic of the proposed opioid receptor subtypes (Kieffer and Gaveriaux-Ruff, 2002). However, we did observe sex differences in the MOR and total opioid receptor density in the cortex of monkeys. There was a significantly greater MOR density and higher degree of DAMGO-stimulated [³⁵S]GTPS binding in the cortex of male monkeys. This finding needs further investigation using more subjects to make potential correlation with MOR-mediated functional significance and to provide an understanding of reported differences in female and male primates to the antinociceptive actions of MOR agonists (Negus and Mello, 1999; Zubieta et al., 2002).

The magnitude of [³⁵S]GTPS binding stimulation by the three selective opioid agonists was similar in cortical membranes. In contrast, different amounts of [³⁵S]GTPS binding were maximally stimulated by the same agonists in thalamic membranes. The magnitude of agonist-stimulated G protein activation was proportional to the corresponding receptor densities in both brain regions. Several in vitro studies have indicated that the relationship between receptor occupancy and G protein activation depends on the receptor density (Selley et al., 1998; Sim-Selley et al., 1999; Maher et al., 2000). DAMGO, U69593 [GenBank] , and SNC80 have been characterized as full MOR, KOR, and DOR agonists, respectively, in the cell lines expressing the corresponding receptors (Clark et al., 1997; Alt et al., 1998; Remmers et al., 1999). The rank order of potencies of these agonists in monkey cortex [U69593 (80 nM) SNC80 (68 nM) > DAMGO (532 nM)] is similar to findings in cell membranes expressing a single opioid receptor type [i.e., U69593 [GenBank] (55 nM) SNC80 (57 nM) > DAMGO (145 nM)] (Clark et al., 1997; Alt et al., 1998; Remmers et al., 1999).

The receptor selectivity of the opioid agonists used in both cortex and thalamus was assured by use of selective opioid antagonists (Clark et al., 1997; Alt et al., 1998; Remmers et al., 1999). The potencies of opioid antagonists used in this study are similar to those in other studies using cell membranes (Alt et al., 1998; Remmers et al., 1999). As noted, the concentration of each antagonist was selective for corresponding receptor antagonism because it did not produce a significant rightward shift of concentration-response curves by other agonists (data not shown). However, the ability of these opioid agonists to activate G proteins could be compromised in brain regions with low receptor density. For example, the full DOR agonist SNC80 was not able to stimulate [³⁵S]GTPS binding in the monkey thalamus, which has relatively low DOR density. A similar finding with the full MOR agonist DAMGO has been reported in specific brain regions of rats (Maher et al., 2000). By using a simple membrane preparation, the present results distinguish the ability of opioid agonists to activate G proteins and provide a functional correlate of ligand-binding experiments in the monkey brain. Moreover, these results support the finding that MOR-stimulated [³⁵S]GTPS binding predominates over KOR-stimulated [³⁵S]GTPS binding in the thalamus of monkeys using [³⁵S]GTPS autoradiography (Sim-Selley et al., 1999). The stimulation of [³⁵S]GTPS binding in both cortex and thalamus is lower than that reported in rat brain membranes (Selley et al., 1998; Fabian et al., 2002), but is in agreement with findings from cynomolgus monkeys (Sim-Selley et al., 1999). Maher et al. (2000) have reported a wide variation in the maximal level of [³⁵S]GTPS stimulation across different regions of rat brain after MOR stimulation. It is likely that differences are due to a combination of receptor density and the concentration of G proteins available for activation. Nevertheless, in spite of the apparently lower level of [³⁵S]GTPS activation, MOR agonists are very effective in the monkey (Butelman et al., 1993; Negus and Mello, 1999; Allen et al., 2002).

Functional imaging studies have revealed the involvement of cortical and thalamic regions in pain perception in humans (Davis et al., 1998; Casey et al., 2000; Peyron et al., 2000; Zubieta et al., 2001). Nevertheless, the thalamus seems to be more involved than the cortex in the modulation of nociceptive processing. First, it has been shown that activities of thalamic neurons, specifically those associated with the transmission of nociceptive signals, can be inhibited by direct or systemic administration of opioids (Nakahama et al., 1981; Brunton and Charpak, 1998). Second, neurological evidence confirms the existence of a highly diverse supraspinal mechanism engaged in the processing of nociceptive intensity. The capacity to evaluate pain intensity is almost completely reserved after extensive cerebral cortical lesions (Knecht et al., 1996; Coghill et al., 1999). Finally, thalamic stimulation used to treat chronic pain in humans for more than two decades has been shown to activate thalamocortical circuits (Duncan et al., 1998).

The different receptor densities and ability to activate G proteins of MOR, KOR, and DOR in the monkey thalamus may be one of several factors contributing to the differential effectiveness of MOR, KOR, and DOR agonists as antinociceptive agents in primates (Negus et al., 1994; Ko et al., 1998; Allen et al., 2002). It is worth noting that in the spinal cord of monkeys, MOR, KOR, and DOR are approximately 68, 25, and 7% of the total binding sites, respectively, and that SNC80 is not able to stimulate [³⁵S]GTPS binding in this region (Lee et al., 2002). The thalamus is the major neural substrate modulating nociceptive signals from both spinothalamic and thalamocortical pathways (Bushnell et al., 1993; Craig et al., 1994; Shi and Apkarian, 1995; Rausell et al., 1998). Thus, it is highly possible that DOR agonists act as weak antinociceptive agents in monkeys due to the lower DOR density and consequent limited G protein activation in the spinothalamic pathway. Our preliminary data showing that intrathecal administration of SNC80 does not produce antinociception against acute thermal stimulus in monkeys (M. C. H. Ko, H. Lee, J. R. Traynor, J. H. Woods, and N. N. Naughton, unpublished observations) supports this notion.

In summary, this study demonstrates the feasibility of using washed tissue homogenates to examine G protein activation in the monkey brain. Results indicate that opioid agonists are able to stimulate [³⁵S]GTPS binding to membranes and that this is a concentration-dependent and receptor-mediated event. More importantly, the relative density of opioid receptor binding sites in particular brain areas may reflect their functional role in the central actions of opioids. Future studies characterizing and comparing the G protein activation by both endogenous and exogenous opioids in the monkey brain will provide much information as to how opioids act in the CNS of primate species.

	Acknowledgements

 
We thank John Busenbark, Tristan Edwards, and Noreen Hughes for excellent technical assistance.

	Footnotes

 
This study was supported by U.S. Public Health Service Grants DA-13685 (to M.C.H.K.) and DA-00254 (to J.H.W.). Preliminary results were presented at the XIVth World Congress of Pharmacology, San Francisco, California, July 7–12, 2002 [Pharmacologist 44 (Suppl):A134].

DOI: 10.1124/jpet.103.050625.

ABBREVIATIONS. MOR, µ-opioid receptor; KOR, -opioid receptor; DOR, -opioid receptor; CNS, central nervous system; GTPS, guanosine-5'-O-(3-thio)triphosphate; DAMGO, [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin; DPDPE, [D-Pen²,D-Pen⁵]-enkephalin; nor-BNI, nor-binaltorphimine; ANVOA, analysis of variance; U69593 [GenBank] , (5,7,8)-(–)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl) benzeneacetamide; SNC80, (+)-4-[(R)--((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxy-benzyl]-N,N-diethyl-benzamide.

Address correspondence to: Dr. M. C. Holden Ko, Department of Pharmacology, University of Michigan Medical School, 1301 MSRB III, Ann Arbor, MI 48109-0632. E-mail: mko{at}umich.edu

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