1 Introduction
Activation of opioid and α2-adrenergic receptor (α2AR) can both inhibit the perception and reaction to painful stimuli. The three cloned opioid receptors (μ, δ, κ) display unique tissue distribution patterns and distinct pharmacological profiles (Heyman et al., 1987; Portoghese et al., 1988; Qiu et al., 2000; Tseng et al., 2000). Currently, most opiate analgesics used clinically act through the μ opioid receptor (MOR), but their use is limited by side effects such as respiratory depression, constipation, dependence and tolerance. The δ opioid receptor (DOR) represents an attractive _target for the development of new drugs to control pain (Quock et al., 1999; Rapaka and Porreca, 1991). Several studies show that some DOR agonists may offer improved therapeutic effects with reduced side effect for clinical treatment of pain (Stimmel, 1989). However, a functional and/or physical interaction between DOR and MOR has been previously proposed (Horan et al., 1992; Jiang et al., 1990; Schoffelmeer et al., 1990). Therefore, further information regarding the involvement of MOR in DOR agonist-induced antinociception is needed.
Agonists and antagonists that have been determined to be selective for one opioid receptor subtype vs. another through in vitro binding assays may exert their effects less selectively when delivered in vivo (Kieffer, 1999; Sofuoglu et al., 1991). The development of knockout (KO) mice with selective deletions of opioid receptor subtypes provides an opportunity to evaluate the effects of drugs in the absence of receptor rather than in the presence of receptor blockade by pharmacological agents, the selectivity of which may be questionable. Two agonists commonly used to evaluate DOR function include the agonists, [d-Pen2,d-Pen5]-enkehphalin (DPDPE, Mosberg et al., 1983) and deltorphin II (DELT II, Melchiorri et al., 1992). Previous studies in MOR-KO mice revealed that in certain antinociceptive assays, supraspinally administered DPDPE and/or DELT II showed decreased antinociception efficacy relative to WT. More specifically, supraspinal and spinal administration of DPDPE (but not DELT II) showed decreased antinociception potency in a line of MOR-KO mice (Hosohata et al., 2000; Sora et al., 1997a,b; exon 1 deletion). In a second line of MOR-KO mice, supraspinal administration of both DPDPE and DELT II showed decreased efficacy in MOR-KO compared to wildtype mice (WT, Matthes et al., 1996, exon 2 disruption). In this present study, we found that spinal administration of DPDPE and DELT II showed decreased antinociceptive potency in a third line of MOR-KO (Loh et al., 1998) compared to WT mice. These convergent studies raise the possibility that antinociception induced by DPDPE or DELT II may be mediated by a direct action at MOR or an enhancement of DOR activity by MOR, such as has been previously proposed (Horan et al., 1992; Jiang et al., 1990; Schoffelmeer et al., 1990).
The present experiments extended the study of DOR agonist-mediated antinociception in MOR-KO mice (exon 2, 3 deletion (Loh et al., 1998)), to include further evaluation of the role of DOR in spinal antinociception. Since the synergistic antinociceptive relationship between the DOR agonist, DPDPE and α2AR-selective agonists has been extensively studied (Fairbanks et al., 2002; Ossipov et al., 1990a,b,c; Roerig et al., 1992; Stone et al., 1997) and presumed to be indicative of DOR–α2AR interactions, it was of particular interest to determine whether MOR participates in those interactions. Therefore, we evaluated the interaction of a DOR–α2AR agonist combination in WT and MOR-KO mice to determine whether DOR synergizes with α2AR when MOR is not present. The results indicate that, in order to achieve full antinociceptive potency, both DPDPE and DELT II require activation of both MOR and DOR. However, DOR–α2AR antinociceptive synergism can be achieved in the ‘absence’ of MOR.
2 Materials and methods
2.1 Animals
Subjects were housed in groups of five in a temperature- and humidity-controlled environment on a 12h light/dark cycle and had free access to food and water. MOR-KO mice were generated as previously described (Loh et al., 1998) using a C57BL/6 and 129/Ola mixed genetic background. WT C57BL/6–129 mice were used as control animals. Within each experiment, the animals were age- and gender-matched across all groups, and they were used between 6 and 8 weeks of age. Each animal was used only once. All experiments were approved by the Institutional Animal Care and Use Committee of the University of Minnesota.
2.2 Chemicals
Substance P (SP) was purchased from Sigma (St. Louis, MO); DPDPE, DELT II, d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Phe-Thr-NH2 (CTOP) and morphine sulfate were provided by the National Institute on Drug Abuse (Rockville, MD). Naltrindole HCl (naltrindole) was a gift from Dr Philip Portoghese (Department of Medicinal Chemistry, University of Minnesota), UK14,304 was a gift from Pfizer. All drugs and peptides were dissolved in 0.9% NaCl and administered intrathecally (i.t.) in a total volume of 5μl according to the method of Hylden and Wilcox (1980).
2.3 Antinociceptive testing
Nociceptive responsiveness was tested using the SP nociceptive test (DeLander and Wahl, 1989; Fairbanks et al., 2002; Fairbanks and Wilcox, 1999; Hylden and Wilcox, 1983a,b; Inoue et al., 1999; Stone et al., 1997). A constant dose (15ng) of SP was administered i.t. in a volume of 5μl, and the number of caudally directed biting, licking and scratching behaviors were counted for 1min after the injection as described previously (Hylden and Wilcox, 1983). SP-induced behaviors have been previously shown to be reversed by opioid and α2AR agonists in a naloxone- and idazoxan-sensitive manner, respectively (Roerig et al., 1992). Furthermore, the correspondence between results using SP and standard antinociceptive tests (e.g. tail flick, Fairbanks and Wilcox, 1999; Stone et al., 1997) support the utility of the SP assay for antinociceptive inference. Since mice with the genetic background C57BL/6–129sv have previously demonstrated sensitive responsiveness to SP (Fairbanks et al., 2002; Fairbanks and Wilcox, 1999; Stone et al., 1997) and since it has been reported that the C57BL/6 strain is less sensitive to thermal stimulation (Mogil and Grisel, 1998; Mogil et al., 1999), we chose to use this test to address the questions posed in this study.
For each experimental day, a new control count was obtained both for WT and MOR-KO mice and the percentage of inhibition was determined relative to the control for each respective mouse line. Control counts ranged from approximately 40 to 60 behaviors in the first minute after injection. A minimum of six mice (three females and three males) were used for each drug or combination dose. To assess the effect of the agonists, each of them was co-administered with SP and inhibition was expressed as a percentage of the mean response of the control group according to the following equation:
2.4 Antagonism study
To identify the receptor requirements for DPDPE-mediated inhibition of SP behavior in WT and MOR-KO, we evaluated the abilities of the MOR-selective antagonist CTOP and the DOR-selective antagonist naltrindole to reverse the effects of DPDPE in both lines of mice. Different doses of DPDPE were co-administered with CTOP (0.01nmol) or naltrindole (8.8nmol). Previous reports, from various other studies, provided guidance in the selection of the doses of CTOP (Tseng and Collins, 1992; Xu et al., 1992) and naltrindole (He and Lee, 1998; Heyman et al., 1987; Portoghese et al., 1988; Tseng and Collins, 1992).
2.5 Statistical analysis
Data describing antinociception are expressed as mean of percent inhibition with SEM. Potency changes are presented as dose ratios between the 50% effective dose (ED50) values of different dose-response curves. The ED50 values, confidence limits (CL), and potency comparisons were calculated according to the method of Tallarida and Murray (1987). For each agonist, a dose–response curve was generated both in WT and MOR-KO lines on the same day.
2.6 Isobolographic analysis
To test for drug interactions, isobolographic analysis was conducted (Tallarida, 1992, 2001, 2002). When testing an interaction between two drugs given in combination for synergy, additivity, or subadditivity, a theoretical additive ED50 is calculated for the combination based on the dose–response curves of each drug administered separately. This theoretical value is then compared by a t-test (P<0.05) with the observed experimental ED50 for the combination of DPDPE and UK14,304. These values are based on total dose of both drugs; in other words, the total dose of DPDPE plus the total dose of UK14,304. An interaction is considered synergistic if the observed ED50 is significantly less (P<0.05) than the calculated theoretical additive ED50. Additivity is indicated when the theoretical and experimental ED50 values do not differ.
3 Results
3.1 SP-elicited behavior in WT and MOR-KO
The number of stereotypical scratching and biting behaviors following intrathecal administration of SP (15ng) did not differ between WT (48±2.2 behaviors, n=46) and MOR-KO (53±2.9 behaviors, n=40, Student's t-test, P>0.05). This result confirms that the behavioral measure was not confounded by the loss of MOR. The number of behaviors observed in response to SP injection did not differ between males and females in either WT (male: 46±2.7, n=26; female: 45±3.2, n=26, Student's t-test, P>0.05) or MOR-KO (male: 55±3.8, n=22; female: 51±3.7, n=24, Student's t-test, P>0.05). This result indicated that the response to the stimulus measured was not confounded by gender and validated the inclusion of both genders in the experimental groups.
3.2 Morphine does not produce antinociception in MOR-KO
To examine the spinal action of morphine in this specific line of MOR-KO mice, we tested morphine for inhibition of SP-evoked behavior. Intrathecally administered morphine dose-dependently produced antinociception in WT, but not in MOR-KO mice, even at the highest dose that could be given without inducing toxicity (Fig. 1, Table 1). This result is consistent with the previous reports of ablation of antinociception induced by intracerebroventricular (ICV) morphine in other lines of MOR-KO mice (Kieffer, 1999; Matthes et al., 1996; Sora et al., 1997a,b). Although there is an ‘apparent’ increase in the MOR-KO mice in the number of SP responses at the 1nmol dose, the number of responses observed in the presence of 1 (70±6.8, n=8), 10 (64±7.1, n=8), or 100 (61±5.0, n=8) nmol morphine is not statistically different from the SP mean baseline in control animals from that experimental day (62±6.2, n=8).
Fig. 1:
Effects of morphine in WT and MOR-KO. Dose–response curves of the spinal antinociceptive effect of morphine in WT (circles) and MOR-KO mice (triangles).
Table 1: Summary of agonist potencies in MOR-WT and MOR-KO mice
3.3 The spinal antinociceptive potency of DOR agonists is decreased in MOR-KO
To examine the spinal action of DOR-selective agonists in this specific line of MOR-KO mice, we tested DELT II and DPDPE for inhibition of SP-evoked behavior. Intrathecally administered DELT II and DPDPE produced antinociception dose-dependently in WT mice. However, in MOR-KO mice, the potency of DELT II was decreased 16-fold and the potency of DPDPE was decreased 250-fold (Fig. 2, Table 1).
Fig. 2:
Effects of DOR-selective agonists in WT and MOR-KO. (A) Dose–response curves of the spinal antinociceptive effect of DPDPE in WT (circles) and MOR-KO (triangles) mice. (B) Dose–response curves of the spinal antinociceptive effect of DELT II in WT (circles) and MOR-KO (triangles) mice.
3.4 DPDPE produces antinociception by acting on DOR and MOR in WT
To determine the receptor requirements for DPDPE-mediated spinal antinociception in WT and MOR-KO mice, we used the MOR-selective antagonist, CTOP (0.01nmol) and the DOR-selective antagonist, naltrindole (8.8nmol), respectively. In WT mice, the co-administration of either CTOP or naltrindole with DPDPE decreased DPDPE antinociceptive potency 27- and 21-fold, respectively (Fig. 3A, Table 1). These observations suggest that in WT mice, DPDPE requires both MOR and DOR activation to achieve full potency.
Fig. 3:
Effects of opioid antagonists on DPDPE-mediated antinociception in WT and MOR-KO. (A) WT mice: dose–response curves of the spinal antinociceptive effect of DPDPE when given alone (circles) and in combination with CTOP (open triangles) or naltrindole (closed triangles). (B) MOR-KO mice: dose–response curves of the spinal antinociceptive effect of DPDPE when given alone (circles) and in combination with CTOP (open triangles) or naltrindole (closed triangles).
3.5 DPDPE produces antinociception by acting on DOR in MOR-KO
In MOR-KO, CTOP did not alter DPDPE potency, however, naltrindole completely ablated DPDPE efficacy (Fig. 3B and Table 1). These observations suggest that in MOR-KO mice, DPDPE-mediated antinociception requires activation of DOR.
3.6 α2AR activation produces antinociception in MOR-KO
Although the WT share the same genetic background as the MOR-KO mice, it could be argued that the absence of morphine antinociception and substantial decrease in DPDPE potency in the MOR-KO mice could be attributable to an overall insensitivity to analgesics in this MOR-KO line. To address that possibility, we compared antinociception produced by a second analgesic class of agonists in the MOR-KO and WT. We tested UK14,304, an α2AR-selective agonist, which has been shown to inhibit SP-evoked behavior in another mixed strain of mice (Stone et al., 1997). Intrathecally administered UK14,304 dose-dependently inhibited SP-evoked behavior in WT and MOR-KO mice with no significant difference in potency between the two lines (Fig. 4A, C, Tables 2 and 3). This result confirms that analgesic sensitivity is retained in the MOR-KO.
Fig. 4:
DOR–α2AR synergy in WT and MOR-KO mice. (A) WT: dose–response curves of the spinal antinociceptive effects of DPDPE (open triangles) and UK14,304 (open circles). For the purpose of comparison to the drug doses administered separately, we have separated the DPDPE and UK14,304 components of the combination dose–response curve and plotted them as two separate dose–response curves: DPDPE in the presence of UK14,304 (closed triangles) and UK14,304 in the presence of DPDPE (closed circles). Drug interactions may also be illustrated through construction of isobolograms, such as are represented in (B) and (D), in these graphs, the ED50 of DPDPE and UK14,304 are, respectively, plotted as the x- and y-axis intercepts. The thicker lines directed from each ED50 value toward 0 represent the respective lower confidence limits of each ED50. The straight line connecting these two points is the theoretical additive line. The open circle that lies on or near the theoretical additive line represents the calculated theoretical ED50 of the combination where the interaction is merely additive. The closed circle represents the experimentally observed ED50 of the combination of DPDPE-UK14,304. If the interaction is synergistic, the closed circle will be plotted significantly below the theoretical additive line and outside the lower confidence limits of ED50 of UK14,304 and DPDPE. (B) Isobolographic analysis was applied to the data from (A) representing combination of DPDPE-UK14,304 in WT, the ED50 of this combination fell below the theoretical additive line and differs significantly from that of the theoretical ED50; therefore, this combination is ‘synergistic’. (C) MOR-KO: dose–response curves of the spinal effect of DPDPE (open triangles), UK14,304 (open circles) and DPDPE in the presence of UK14,304 (closed triangles) and UK14,304 in the presence of DPDPE (closed circles). (D) Isobolographic analysis was applied to the data from (C) that represent the combination of DPDPE-UK14,304 in MOR-KO. The ED50 value of this combination falls below the theoretical additive line and differs significantly from that of the theoretical ED50; therefore, this combination is ‘synergistic’.
Table 2: Summary of DPDPE-UK14,304 spinal antinociceptive synergy in MOR-WT mice
Table 3: Summary of DPDPE-UK14,304 spinal antinociceptive synergy in MOR-KO mice
3.7 Synergy between DPDPE and UK14,304 in WT and MOR-KO
The phenomenon that DPDPE and DELT II both require MOR to achieve full potency in the WT raises the question of whether or not these DOR agonists also require MOR to participate in their synergism with α2AR-selective agonists. The observation that the potency of the α2AR-selective agonist UK14,304 was not altered in MOR-KO enabled this evaluation. Since DPDPE showed the greatest requirement for MOR activation, we chose this agonist for the synergism studies. Our rationale was that if MOR is required for DPDPE-UK14,304 synergism, that interaction will be reduced to additivity in MOR-KO.
In WT mice, the DPDPE-UK14,304 equi-effective dose ratio administered was 1.35:1. When administered in combination, the ED50 values of DPDPE and UK14,304 were markedly lower than that of either agonist administered individually (Fig. 4A, Table 2). Therefore, the potency of DPDPE substantially increased in the presence of UK14,304, and likewise the potency of UK14,304 markedly increased in the presence of DPDPE. Isobolographic analysis revealed that the observed ED50 value of the combination was significantly less than the calculated theoretical additive ED50 (Fig. 4B, Table 2). This result confirms a synergistic interaction between DPDPE-UK14,304.
In KO mice, the DPDPE-UK14,304 equi-effective dose ratio administered was 100:1. When administered in combination, the ED50 of DPDPE and UK14,304 are also markedly lower than that of either agonist administered individually (Fig. 4C, Table 3). This observation indicates an increase in potency for each drug administered in the presence of the other compared to each drug administered alone. Isobolographic analysis revealed that the observed ED50 of the combination was significantly less than the calculated theoretical additive ED50 value (Fig. 4D, Table 3). This result confirms a synergistic interaction between DPDPE-UK14,304 in the absence of MOR. It suggests that DOR activation is sufficient (MOR is not required) for DPDPE-UK14,304 antinociceptive synergy.
4 Discussion
The present study demonstrates that the DOR-selective agonists DPDPE and DELT II, require both MOR and DOR for full spinal antinociceptive potency. These results that are derived from genetic-induced ablation of MOR combined with pharmacological blockade of MOR and DOR are consistent with a previous pharmacological study by He and Lee (1998) where it was demonstrated that the MOR-selective antagonist, CTAP (d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2) antagonized both DAMGO ([d-Ala 2, N-MePhe4, Gly5–01]-enkephalin)- and DPDPE-induced spinal antinociception. These results are also consistent with the reports from two other lines of MOR-KO mice showing decreases in the antinociceptive efficacy of DPDPE and/or DELT II (Hosohata et al., 2000; Matthes et al., 1998; Sora et al., 1997a,b). Finally, the present study showed that a DOR-selective agonist (DPDPE) with a strong MOR dependence for full spinal antinociceptive potency activates DOR for synergistic interaction with α2AR independent of MOR.
4.1 Differential decrease in DPDPE and DELT II potency in MOR-KO
We observed a decrease in potency of DPDPE and DELT II in MOR-KO mice. These results are comparable to observations reported in a second line of MOR-KO mice (Hosohata et al., 2000), where significant decreases in spinal DPDPE potency in MOR-KO mice (9-fold); however, a change in spinal DELT II-mediated antinociception was not detected in that study. The different tests of nociception used between the two studies (present study: SP test; Hosohata study: tail flick 55°C test) could account for the difference. We observed a 250-fold whereas Hosohata et al. (2000) observed a ninefold decrease in DPDPE potency. Similarly, we observed a 16-fold decrease whereas Hosohata and colleagues observed no change in DELT II potency; consequently, the SP test may provide greater sensitivity for detection of small effects. Most importantly, both studies agree that DPDPE-mediated antinociception has a greater MOR dependence than DELT II.
4.2 Synergy between DPDPE and UK14,304 in MOR-KO
When agonists to α2AR and opioid receptors are co-administered with SP, they act synergistically to inhibit SP-elicited behavior (Hylden and Wilcox, 1983; Roerig et al., 1992). Previous works have suggested that activation of spinal DOR participates in such interactions (Roerig et al., 1992; Stone et al., 1997; Fairbanks et al., 2002). However, those previous studies have relied upon the use of DOR agonists, DPDPE and DELT II, which have now been shown to recruit the participation of MOR. It is possible, therefore, that MOR is involved in the synergistic interaction with the α2AR agonists. Having established that DOR function is retained in MOR-KO from studies of binding, distribution, and G-protein activation or peptide message level (Qiu et al., 2000), and that α2AR-mediated antinociception is unchanged in MOR-KO (present study), we took the opportunity to evaluate DOR–α2AR antinociceptive interactions in the absence of MOR. To evaluate the role of DOR in DOR–α2AR synergy, we elected to use DPDPE, rather than DELT II because DPDPE showed a greater dependence on MOR than did DELT II. We reasoned that increased dependence of MOR would make it more difficult for DPDPE to synergize with an α2AR agonist in MOR-KO, making the use of this particular DOR agonist a more conservative test of the hypothesis.
In a previous study, evaluation of opioid–α2AR interactions in a mouse line deficient in the α2AAR subtype showed that the spinal antinociceptive potency of UK14,304 was reduced 250-fold and that the DPDPE-UK14,304 synergy observed in the WT was reduced to additivity in the mutant mice (Stone et al., 1997). This result demonstrated that the deficient receptor in those studies was required for both full agonist potency and synergy with opioid agonists. In contrast, we observed DPDPE-UK14,304 antinociceptive synergy in both WT and MOR-KO, despite the 250-fold decrease in DPDPE potency in MOR-KO. The reduction of the DPDPE-UK14,304 synergy to additivity in α2AAR mutant mice, but persistence in MOR-KO, underscores the importance of DOR in antinociception produced by this agonist combination.
4.3 DOR in DPDPE- and DELT II-mediated antinociception and synergy effect
The persistence of DPDPE-UK14,304 synergy in MOR-KO supports a spinal antinociceptive role for DPDPE independent of MOR. These data indicate that, in the absence of MOR, this DOR-selective agonist can produce antinociception mediated by DOR activation. Furthermore, when we evaluated the effects of the DOR antagonist naltrindole on DPDPE-mediated antinociception, we observed that naltrindole completely prevented DPDPE mediated-antinociception in MOR-KO as well as demonstrated a decrease in DPDPE potency in WT. These results show that DPDPE does, in fact, act directly on DOR to produce antinociception in the absence of MOR.
4.4 Interactions between DOR and MOR
From our results presented above, we conclude that spinally administered DPDPE is less selective for DOR in vivo than previously thought, but we cannot exclude the possibilities that there are interactions between MOR and DOR. Several lines of evidence have been suggested that DOR and MOR interact in spinal antinociception. For example, DOR and MOR agonists have been shown to interact in a synergistic manner (Jiang et al., 1990; Malmberg and Yaksh, 1992; Schoffelmeer et al., 1990; Traynor and Elliott, 1993). Other studies have reported that DOR antagonists can modulate MOR-mediated antinociception (Malmberg and Yaksh, 1992). As stated previously, the three separate lines of MOR-KO mice show lack of response to morphine, suggesting that morphine is a MOR-selective agonist in vivo. However, other reports have shown that DOR activation mediates morphine-induced inhibition of met-enkephalin release in rat spinal cord (Collin et al., 1994), that DOR participates in the development of morphine dependence and tolerance in mice (Miyamoto et al., 1993), and that DOR activation enhances morphine's antinociception observed under inflammatory conditions (Ossipov et al., 1995). Whether the interaction between MOR and DOR relies on a distant interaction with a functional outcome or an actual physical association, remains to be determined.
Based on recent anatomical and biochemical reports, we can speculate on several possible sources of interaction. First, immunoreactivity for MOR and DOR have different patterns of localization in the spinal cord (Arvidsson et al., 1995a,b); MOR resides in both primary afferent terminals and in second order spinal neurons whereas DOR is found almost exclusively on the terminals of primary afferents (Arvidsson et al., 1995a,b; Dado et al., 1993). Direct activation of both receptors by less selective agonists (e.g. DPDPE) could (1) inhibit neurotransmitter release from the DOR- and/or MOR-containing primary afferent terminal and (2) hyperpolarize second-order MOR-containing neurons preventing neuronal transmission. Through these two actions, an agonist activating both receptors could thereby produce synergistic effects in series in a manner similar to that observed when DOR and MOR agonists are co-administered.
Second, recent biochemical evidence suggests that G-protein-coupled opioid receptors pairs KOR–DOR (Jordan and Devi, 1999) and MOR–DOR (Gomes et al., 2000) form heterodimers in vitro. It is, therefore, conceivable that MOR and DOR form heterodimers in vivo and that the spinal antinociception mediated by DPDPE and/or DELT II activates such heterodimers, perhaps even MOR–DOR1 and MOR–DOR2, respectively. Anatomical evidence demonstrating that many primary afferents are positive for both MOR and DOR mRNA indicates that DOR–MOR dimers are possible (Wang and Wessendorf, 2001). However, subcellular localization studies using electron microscopy suggest that co-localization of DOR and MOR on the same subcellular compartment is rare, making a direct physical interaction unlikely (Cheng et al., 1997).
5 Conclusion
The present report extends the study of MOR participation in DOR agonist-mediated antinociception to include a spinal site in a third MOR-KO mouse line (exon 2, 3 deletion). In these mice, the potency of intrathecally administered DELT II and DPDPE decreased, relative to WT controls. That these results parallel similar findings reported in two other independently generated lines of MOR-KO strongly supports a role for MOR in DPDPE- and DELT II-mediated antinociception. However, the present report's observation that DPDPE synergizes with UK14,304 in MOR-KO demonstrates that, while DPDPE shows significantly greater potency with activation of ‘both’ MOR and DOR, activation of DOR (in the absence of MOR) is sufficient to interact synergistically with another G-protein-coupled receptor, α2AR.
Acknowledgements
We extend our great appreciation to Drs George L. Wilcox and Ping-Yee Law, Tinna M. Laughlin, Rachid E.L. Kouhen and Smita Kshirsagar for useful conversations; to Dr Michael H. Ossipov for computational assistance with isobolographic analysis; to Mr Kelley F. Kitto, Ms H. Oanh Nguyen, Mr Tyler Crockett and Ms Lori Kaminski for excellent technical assistance. Supported by DA-00546, DA-01583 and by the A.F. Stark Fund of the Minnesota Medical Foundation (HHL); NCCAM training grant P50AT00009–02 and DA-00509–01A1 supported C.A.F.
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