Introduction

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Opioid receptors are expressed throughout the central nervous system and are believed to modulate a variety of behavioral responses, including antinocicieption, mood, dependence, motivation, and depression (Dhawan et al., 1996). Three opioid receptor subtype genes (, µ, ) have been cloned to date (Evans et al., 1992; Kieffer et al., 1992; Chen et al., 1993; Yasuda et al., 1993) and further receptor heterogeneity for all three classes of opioid receptors has been proposed (Dhawan et al., 1996). Common analgesics such as morphine and related compounds preferentially interact with the µ-opioid receptor subtype (Pasternak, 1993). However, the therapeutic benefit of µ-opioid receptor agonists is diminished by the appearance of side effects, including dependence, constipation, and respiratory depression (Pasternak, 1993). Consequently, the therapeutic potential of agonists selective for other opioid receptors is under investigation. In this context, -agonists are of particular interest because they mediate antinociception in laboratory animals yet produce fewer adverse effects than µ-agonists (Quock et al., 1999).

-Opioid receptors have been proposed to exist in two pharmacologically distinct subtypes, the evidence being based in large part on comparisons between the prototypical agonists deltorphin II and DPDPE. Thus, deltorphin II and DPDPE-mediated adenylyl cyclase stimulation in rat brain preparations (Búzás et al., 1994; Olianas and Onali, 1995) as well as antinociception in both rats (Thorat and Hammond, 1997) and mice (Jiang et al., 1991; Sofuoglu et al., 1991; Vanderah et al., 1994) was differentially antagonized by various -antagonists. In addition, cross-tolerance in mice was not observed between the antinociceptive effects of DPDPE and deltorphin II, or with either of these peptides and the µ-agonist DAMGO (Mattia et al., 1991). In total, these studies provide strong evidence that DPDPE and deltorphin II interact with distinct sites. However, the determination of the identity and function of these unique sites is complicated by the heterogeneous population of opioid receptors expressed in tissues such as brain (Mansour et al., 1995) and the limited selectivity of the pharmacological tools used to resolve individual sites.

Antisense and genetic knockout approaches provide powerful alternative methods for the determination of receptor function (Fraser and Wahlestedt, 1997). Antisense studies performed in mice support the existence of -receptor subtypes mediating antinociception in the brain and further suggest that these subtypes may arise from splice variants of the cloned -opioid receptor (DOR) gene (Rossi et al., 1997). In contrast, supraspinal antinociception in response to -agonists, including DPDPE and deltorphin II, is reported to persist in DOR knockout mice (Zhu et al., 1999). The latter observation implies that certain -agonists interact with receptors other than DOR in the mouse brain, a finding that calls into question the role of DOR in mediating supraspinal antinociception.

The primary objective of the present study was to re-evaluate the role of DOR in the modulation of supraspinal antinociception in the rat. A second objective was to investigate discrepancies in the pharmacology of common -agonists with application to the possible existence of -opioid receptor subtypes.

	Materials and Methods

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Animals. Male Sprague-Dawley rats (250-300 g; Charles River, St-Constant, Québec, Canada) were housed in groups of three under an artificial 12-h light/dark cycle in a climate-controlled environment (23°C, relative humidity 60%). Food and water were provided ad libitum to animals throughout the housing period. Animals were used in compliance with the guidelines established by the Canadian Council for Animal Care.

Surgery. Rats were anesthetized by intraperitoneal injection of ketamine (80 mg/kg)/xylazine (12 mg/kg) solution (Research Biochemicals International, Natick, MA) and placed in a stereotaxic device aligned with the interaural line. Each animal was implanted with a 23-gauge stainless steel cannula extending into the right lateral ventricle of the brain (i.c.v.; coordinates from bregma, AP, 0.8 mm; ML, 1.5 mm; DV, 3.5 mm). The guide cannula was fixed into place with dental cement applied to the surface of the skull. Rats were allowed 3 to 7 days to recover from surgery before random allocation into treatment groups.

Oligodeoxynucleotides. Phosphodiester antisense and mismatch oligodeoxynucleotides (ODN) were synthesized by Midland Certified Reagent Co. (Midland, TX). The 20-base antisense ODN (5'-GCA CGG GCA GAA GGC AGC GG-3') was designed complementary to nucleotides 112 to 131 (exon 1) of the rat -opioid receptor, a region analogous to the 5' end of the coding sequence previously targeted in mouse (Bilsky et al., 1996). A mismatch sequence (5'-GCA GCG GCA AGA GGA CGC GG-3') comprising the same base composition as the antisense sequence was designed to test the sequence-specificity of the antisense ODN. A search of the GenBank database confirmed that neither ODN sequence was homologous to any known nontarget genes in the rat. ODNs were reconstituted in sterile 0.9% saline solution on the first treatment day and stored at 4°C for the duration of the treatment period. ODNs were administered i.c.v. in bolus injections of 20 µg/10 µl at 12-h intervals for 5 days. Vehicle-treated control subjects were dosed concurrently.

Chemicals. Naloxone and the opioid peptides [D-Ala²,Glu⁴]-deltorphin (deltorphin II), [D-Pen^2,5]-enkephalin (DPDPE), [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (DAMGO), and D-Phe-c[-Cys-Tyr-D-Trp-Orn-Thr-Pen]-Thr-NH₂ (CTOP) were purchased from Research Biochemicals International. [D-Pen²,pCl-Phe⁴,D-Pen⁵]-enkephalin (pCl-DPDPE) was purchased from Bachem (Basel, Switzerland). SNC80 was purchased from Tocris Cookson (Ballwin, MO). All drugs were weighed out and dissolved in 0.9% saline solution [or 10% dimethyl sulfoxide (DMSO) for pCl-DPDPE] immediately before experimentation. The radioligand ¹²⁵I-AR-M100613 (¹²⁵I-Dmt-c[-D-Orn-2-Nal-D-Pro-D-Ala-]), was synthesized in our laboratories as previously described (Fraser et al., 1999).

Intracerebroventricular Injections. ODNs and opioid drugs were administered via the i.c.v. route to conscious rats via the indwelling guide cannula. Injections were made using a 50-µl Hamilton syringe attached via PE20 polyethylene tubing to a 30-gauge injection cannula. Solution was injected over a period of 60 s. The injection cannula was left within the guide cannula for an additional 30 s to minimize reflux.

Antinociceptive Testing. Each rat was tested on only one occasion. The same investigator performed all antinociceptive testing. Acute mechanonociception was measured using an analgesy meter (Ugo Basile, Varese, Italy). Briefly, a rat is gently restrained by hand and an increasing force is gradually applied to the right hind paw at a constant rate until the threshold force causing the rat to withdraw its paw is determined. A maximal cut-off force of 510 g was implemented for this study. Data presented as percentage maximum possible effect (%MPE) were determined using the following calculation: %MPE = [(response  baseline)/(cut-off  baseline)] × 100%.

Radioligand Binding Studies. Antisense, mismatch, and vehicle-treated control rats were decapitated immediately after the hour-long test session. The whole brain (minus cerebellum) was rapidly dissected and stored at 70°C before preparation of membrane homogenates. Brain homogenates were prepared from antisense, mismatch and saline-treated animals administered deltorphin II or DPDPE (n = 4 sets for each -agonist, respectively). On the day of homogenate preparation, brains were thawed and washed in 0.25 mM EDTA/0.5 M phosphate buffer solution (pH 7.4, 4°C) and then individually homogenized in a 20-ml solution of 50 mM Tris buffer, 2.5 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride (pH 7.0). P₂ homogenate fractions were prepared from two consecutive low-speed centrifugation steps (1200g). The resulting supernatant was then centrifuged twice at 48,000g (20 min for each spin) at 4°C. The P₂ pellet was resuspended in 5 ml of 50 mM Tris buffer (pH 7.4) and incubated at 37°C for 15 min to dissociate any receptor-bound endogenous opioid peptides. Membranes were centrifuged a final time at 48,000g and the pellet was resuspended in 5 ml of 50 mM Tris buffer/0.32 M sucrose solution (pH 7.0). Protein content was determined by modified Lowry assay with sodium dodecyl sulfate. Membrane aliquots were rapidly frozen in dry ice/ethanol and stored at 70°C until the day of the binding assay.

Data Analysis. All analyses were performed using Prism (version 2.01) from GraphPad Software (San Diego, CA). Dose-response effects were analyzed by two-way ANOVA with dose and time as between-subject and within-subject factors, respectively. ED₅₀ and ED₈₀ values were determined by linear regression analyses of the dose-response curves. Comparisons between the saline, antisense, and mismatch-treated test groups were made by one-way ANOVA. Post hoc analyses were performed with Dunnett's multiple comparison test or Bonferroni t tests, as appropriate. Receptor binding data were analyzed by nonlinear least-squares regression analysis.

	Results

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Opioid Agonists Modulate Acute Mechanonociception in the Paw Pressure Assay. Dose-response curves were established for the µ-agonist DAMGO, and the putative -agonists deltorphin II, DPDPE, pCl-DPDPE, and SNC80. The different doses of each agonist were tested in parallel in comparison to vehicle-treated controls. Dose-response effects were normalized to the control baseline and data presented as %MPE to facilitate comparison of dose-response curves for agonists tested on different days. All five test compounds gave a similar response profile; antinociception was maximal at the 15-min test interval, and also the 30-min test interval in the case of deltorphin II, but not significant at the 60-min test interval in comparison to saline-treated controls (data not shown). Treatment with each opioid significantly increased response thresholds in a dose-dependent manner (Fig. 1): DAMGO, ED₅₀ = 0.096 nmol, F_4,156 = 36, P < .001; deltorphin II, ED₅₀ = 34 nmol, F_4,184 = 39, P < .001; DPDPE, ED₅₀ = 53 nmol, F_3,124 = 22, P < .001; pCl-DPDPE, ED₅₀ = 100 nmol, F_4,152 = 32, P <. 001; and SNC80, ED₅₀ = 240 nmol, F_4,164 = 25, P < .001. 

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Antinociceptive dose-response curves for DAMGO (), deltorphin II (), DPDPE (), pCl-DPDPE (), and SNC80 () in the paw pressure assay. The data represent the peak antinociceptive effects for each agonist measured at 15 min (or 30 min for deltorphin II) after injection (i.c.v.) for each drug. Data are presented as a percentage of the maximum possible effect (%MPE) that can be measured using this test paradigm. Each data point represents the mean ± S.E.M. response of 8 to 12 rats.

Antisense Inhibition of -Opioid Receptor-Mediated Antinociception. The antinociceptive response to ED₈₀ concentrations of the opioid agonists (derived from the data presented in Fig. 1) was measured in rats pretreated with antisense (or mismatch) oligonucleotides (i.c.v.) targeted against the -opioid receptor in comparison to vehicle-treated controls. As expected, the peak antinociceptive effects for each opioid agonist were observed at the 15- to 30-min test intervals in vehicle-treated subjects. Figure 2, A to E, shows the effects of antisense (and mismatch) treatment on rats administered opioid agonists. Antisense treatment significantly inhibited increases in nociceptive response thresholds in response to SNC80, deltorphin II, and pCl-DPDPE (Fig. 2, A-C, respectively) but not DPDPE or the µ-agonist DAMGO (Fig. 2, D and E, respectively). In comparison, treatment with the mismatch sequence did not significantly alter the antinociceptive response to any of the opioid agonists at any test interval (P > .05). In addition, antisense or mismatch treatment did not significantly alter the baseline nociceptive responses measured for all treatment groups just before the administration of opioid agonists.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Administration of antisense oligonucleotides targeting -opioid receptors inhibited the antinocicieptive response to SNC80 (400 nmol) (A), deltorphin II (60 nmol) (B), and pCl-DPDPE (160 nmol) (C), but not DPDPE (100 nmol) (D) or DAMGO (0.2 nmol) (E). * and ** represent significant differences in comparison to the vehicle + agonist group where P < .05 and 0.01, respectively (Dunnett's t test). Each data point represents the mean ± S.E.M. response of 7 to 11 rats. Veh, vehicle; AS, antisense ODN; MM, mismatch ODN. Control rats were administered saline (i.c.v.) twice daily to simulate the antisense treatment regimen and also administered saline (i.c.v.) to control for drug treatment on the test day.

View this table: [in this window] [in a new window]   TABLE 1 Effect of antisense treatment on  opioid receptor density in whole brain homogenates 125I-AR-M100613 saturation binding was performed on sets of whole brain homogenates from saline-, antisense-, and mismatch-treated rats administered either deltorphin II or DPDPE (n = 4 sets for each -agonist). Binding assays were performed in the presence of 50 nM CTOP to minimize residual binding of the radioligand to µ-opioid receptors. Each homogenate sample was assayed separately. Data are presented as mean ± S.E.M.

Inhibition of Antinociception by the µ-Opioid Antagonist CTOP. Preliminary experiments indicated that 0.5 nmol of CTOP (i.c.v., given 10 min before agonist) was the minimal dose required to completely block the antinociceptive effects of the µ-agonist DAMGO (0.2 nmol i.c.v., data not shown). Figure 3 shows the effects of CTOP (0.5 nmol i.c.v., given 10 min before agonist) on the antinociceptive responses to ED₈₀ concentrations of deltorphin II, SNC80, pCl-DPDPE, DPDPE, and DAMGO (60, 400, 160, 100, and 0.2 nmol i.c.v., respectively; tested 15 min after dosing). This experiment was performed in two parts where deltorphin II, DPDPE, and DAMGO, and then SNC80 and pCl-DPDPE, were tested in parallel alongside vehicle and CTOP-treated controls. The response thresholds from the vehicle and CTOP-treated control subjects did not differ between experiments; these data were pooled and are presented in Fig. 3. Pretreatment with CTOP significantly inhibited the antinociceptive responses to DAMGO and DPDPE (Bonferroni t test: t = 9.58, df = 16, P < .001 and t = 9.03, df = 16, P < .001, respectively). Little if any residual agonist response occurred in the presence of the antagonist. In addition, CTOP inhibited the antinociceptive response to pCl-DPDPE (Bonferroni t test: t = 3.49, df = 12, P < .005), although a significant agonist response occurred in the presence of the antagonist in comparison to CTOP-treated controls (Bonferroni t test: t = 3.69, df = 8, P < .01). In contrast, CTOP did not inhibit the response to deltorphin II or SNC80 (Bonferroni t test: t = 0.89, df = 16, P = .39 and t = 0.92, df = 15, P = .37, respectively), or alter the response threshold in saline-treated controls (Bonferroni t test: t = 1.19, df = 15, P = .25).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Pretreatment with CTOP (0.5 nmol, i.c.v., 10 min before agonist) antagonized the antinociceptive response to DAMGO (0.2 nmol), DPDPE (100 nmol), and pCl-DPDPE (160 nmol), but not deltorphin II (60 nmol) or SNC80 (400 nmol). The figure depicts the antinociceptive response to opioid agonist (i.c.v.) at 15 min post injection. Each column (, +vehicle; , +CTOP) represents the mean ± S.E.M. of six to nine rats. * and ** represent significant differences between the CTOP-treated and untreated groups for each agonist condition, where P < .005 and P < .001, respectively (Bonferroni t test).

	Discussion

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The present study demonstrates that the antinociceptive effects of deltorphin II, SNC80, and pCl-DPDPE, but not DPDPE, were inhibited by antisense treatment targeted against the cloned DOR. Additional studies demonstrated that the antinociceptive response to DPDPE was completely blocked by pretreatment with the selective µ-antagonist CTOP. In total, these findings confirm the role of DOR in the modulation of antinociception at supraspinal sites and further suggest that the pharmacological actions of DPDPE are distinct from those of other -agonists.

Opioid receptors in the brain modulate descending pain pathways and consequently increase nociceptive response thresholds (Basbaum and Fields, 1984). The antinociceptive response to µ-agonists administered into the brain has been clearly demonstrated (Fang et al., 1986). In comparison, in studies performed in rats, -opioid agonists (administered i.c.v.) have been reported to have discrepant effects on nociception that appear to be contingent upon the agonists and the nociceptive assays used (Negri et al., 1991; Adams et al., 1993; Ossipov et al., 1995). The paw pressure assay is more sensitive to the effects of opioids (i.c.v.) than tests measuring spinal reflex responses (Hayes et al., 1987; Miaskowski et al., 1991). The outcome of this nociceptive test, the paw withdrawal response, is an organized, unlearned behavior requiring supraspinal processing (Dubner, 1989). In the present study, all the compounds tested attained maximal efficacy in the paw pressure assay.

It has been suggested that the antinociceptive response to high concentrations of various -agonists may in fact be a consequence of a low-affinity, nonselective direct activation of µ-receptors (Negri et al., 1996). This hypothesis was tested in the present study using antisense and CTOP administration to assess possible DOR and µ-receptor involvement, respectively. Antisense treatment inhibited the antinociceptive response to deltorphin II, pCl-DPDPE, and SNC80 in a sequence-specific and pharmacologically selective manner. The inhibition of response to these agonists was associated with a reduction of -opioid binding sites in brain homogenates prepared from antisense-treated rats. These findings suggest that DOR plays an important role in the modulation of supraspinal pain pathways in the rat, a finding consistent with that of previous antisense studies performed in the mouse (Standifer et al., 1994; Bilsky et al., 1996; Rossi et al., 1997). Moreover, DOR-mediated antinociception is independent of µ-receptor activation based on the inability of the selective µ-antagonist CTOP to inhibit deltorphin II- or SNC80-mediated increases in paw withdrawal latency.

The pharmacology of DPDPE was distinct from that of the other -agonists used in this study, in two respects: insensitivity to antisense treatment and complete antagonism by CTOP. It is unlikely that these findings reflect differences in agonist efficacy between DPDPE and the other -agonists tested because all agonists were used at approximately ED₈₀ concentrations in these experiments. The observed lack of inhibition by the antisense sequence suggests either that DPDPE does not modulate supraspinal nociception exclusively via the DOR receptor or that DPDPE activates an anatomically distinct receptor population that is differentially affected by the antisense treatment. Similar findings have been reported in antisense studies performed in mice (Bilsky et al., 1996; Rossi et al., 1997). In addition, a recent study demonstrates that the effects of DPDPE (i.c.v.), but not deltorphin II, on locomotor activity are resistant to antisense treatment in rats (Negri et al., 1999). The results of these antisense studies may appear to contrast with published reports where -selective antagonists have been found to block the effects of DPDPE (Sofuoglu et al., 1991; Búzás et al., 1994). However, the antisense techniques used in the present study specifically target DOR, whereas the antagonists previously used may inhibit the effects of DPDPE via interactions with a heterogeneous population of sites.

The second distinctive feature of DPDPE antinociception, in comparison to that of other -agonists, was its complete blockade by the µ-selective antagonist CTOP. This finding is in agreement with data presented in previous studies in mice where the antinociceptive effects of DPDPE were blocked by pretreatment with the highly selective µ-antagonist CTAP (D-Phe-c[-Cys-Tyr-D-Trp-Arg-Thr-Pen]-Thr-NH₂) at the level of the brain (Kramer et al., 1989) or spinal cord (He and Lee, 1998). Further support for a µ-component to DPDPE-mediated antinociception has been provided by studies with µ-receptor knockout mice (Sora et al., 1997; Matthes et al., 1998; Fuchs et al., 1999; Hosohata et al., 2000; but see Loh et al., 1998). The present study demonstrates that the µ-dependent effects of DPDPE can occur at doses that are submaximal with respect to antinociception. In comparison, the DPDPE analog pCl-DPDPE appears to mediate supraspinal antinociception via both µ-dependent and -independent sites based on the inhibition of pCl-DPDPE effects by both DOR antisense and CTOP pretreatment.

Several findings suggest that the observed µ-receptor dependence of DPDPE antinociception may reflect, at least in part, a direct interaction of the agonist with supraspinal µ-opioid receptors. For example, DPDPE proved more potent than pCl-DPDPE in the present antinociceptive assay, even though DPDPE has a lower binding affinity for -opioid receptors and a much higher affinity for µ-receptors (Kramer et al., 1993). Also, the greater sensitivity of DPDPE to CTOP inhibition is consistent with its inferior /µ-receptor binding selectivity in comparison to pCl-DPDPE (Kramer et al., 1993). Furthermore, binding studies performed on cell lines expressing recombinant human opioid receptors have revealed only moderate (approximately 100-fold) /µ-selectivity for DPDPE and for the reversible -antagonists reported to block the effects of DPDPE [i.e., naltrindole, BNTX (7-benzylidenenaltrexone), naltriben, ICI174,864, all less than 200-fold /µ-selective; Payza et al., 1996]. Similarly, the irreversible antagonist DALCE ([D-Ala²,Leu⁵,Cys⁶]enkephalin), which blocks certain effects of DPDPE (Jiang et al., 1991; Vanderah et al., 1994), also appears to have some affinity for µ-receptors (Bowen et al., 1987). Thus, in tissues such as brain where µ-receptors are predominant (Mansour et al., 1995), it is conceivable that even low levels of µ-receptor occupancy by DPDPE and by -selective antagonists may be behaviorally significant.

Alternatively, DPDPE may elicit supraspinal antinociception by acting on certain -sites that, in turn, potentiate µ-receptor activity (Traynor and Elliot, 1993). This hypothesis is supported by neuroanatomical studies demonstrating that - and µ-opioid receptors are coexpressed in certain brain regions (Mansour et al., 1995). In addition, previous studies have shown that the coadministration of DPDPE with µ-agonists caused a synergistic increase in supraspinal antinociception (Miaskowski et al., 1991; Negri et al., 1995). Although the nature of this µ/-receptor interaction is unclear at present, it likely does not occur at the level of signal transduction because -agonist-induced G-protein activation or adenylyl cyclase inhibition were not affected in µ-receptor knockout mice (Matthes et al., 1998). Alternatively, pharmacological data supports the existence of a µ/-receptor complex (Rothman et al., 1988; Traynor and Elliot, 1993) such as the recently identified hetero-oligomer formed between DOR and the cloned µ-opioid receptor (George et al., 2000). Nevertheless, the antisense experiments in the present study suggest that any indirect activation of µ-receptors by DPDPE was likely mediated by DOR-independent sites.

The existence of -opioid receptor subtypes has been postulated, in large part, on the basis of differences in the pharmacology of the prototypical -agonists deltorphin II and DPDPE (Jiang et al., 1991; Mattia et al., 1991; Vanderah et al., 1994). However, a second subtype arising from a gene distinct from DOR was not revealed by [³H]DPDPE or [³H]deltorphin II binding in brain homogenates prepared from DOR knockout mice (Zhu et al., 1999). Alternatively, previous antisense studies in mice suggest that splice variants of the common DOR gene may give rise to receptor subtypes (Rossi et al., 1997). The present study demonstrates that DPDPE interacts with a site that is distinct from that targeted by other -agonists; this site is directly or indirectly associated with µ-opioid receptors. Further studies are required to determine whether the DPDPE site is a novel -opioid receptor (possibly arising from a different gene or DOR splice variant) or the µ-opioid receptor.