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NEUROPHARMACOLOGY
-2A- or -2C-Adrenergic ReceptorsFaculty of Dentistry, McGill Centre for Research on Pain, McGill University, Montreal, Quebec, Canada (L.S.S.); Departments of Neuroscience (L.S.S., K.F.K., C.A.F., G.L.W.), Pharmacology (C.A.F., G.L.W.), and Dermatology (G.L.W.), University of Minnesota Medical School, Minneapolis, Minnesota; Department of Pharmaceutics, College of Pharmacy (K.F.K., C.A.F.), University of Minnesota, Minneapolis, Minnesota; Minnesota Center for Research on Pain, Minneapolis, Minnesota (L.S.S., K.F.K., C.A.F., G.L.W.); and Department of Anesthesiology, Wake Forest University Medical Center, Winston-Salem, North Carolina (J.C.E.)
Received July 31, 2007; accepted September 12, 2007.
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Abstract |
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2-adrenergic receptors (2ARs) produce antinociception and synergize with opioids. The 2ARs are divided into three subtypes, 2AAR, 2BAR, and 2CAR. Most 2AR agonists require 2AAR activation to produce antinociception and opioid synergy. The same subtype also mediates the side effect of sedation, which limits the clinical utility of these compounds. Identification of a non-2AAR-mediated antinociceptive agent would enhance the therapeutic utility of 2AR agonists in pain management. Previous studies have suggested that the 2AR agonist ST91 [2-(2,6-diethylphenylamino)-2-imidazoline hydrochloride] has a nonsedating, non-2AAR mechanism of action. We examined the pharmacology of spinal ST91 and its interaction with the 
-opioid agonist deltorphin II (Tyr-D-Ala-Phe-Glu-Val-Val-Gly amide) in mice lacking either functional 2AARs or 2CARs. All drugs were administered by direct lumbar puncture, and drug interactions were evaluated using isobolographic analysis. In contrast to the majority of 2AR agonists, ST91 potency was only moderately reduced (3-fold) in the absence of the 2AAR. Studies with the 2AR subtype-preferring antagonists BRL-44408 (2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidazole maleate) and prazosin [[4-(4-amino-6,7-dimethoxy-quinazolin-2-yl) piperazin-1-yl]-(2-furyl)methanone] and the pan-2AR antagonist SKF-86466 (6-chloro-2,3,4,5-tetrahydro-3-methyl-1-H-3-benzazepine) suggest a shift from 2AAR to the other 2AR subtype(s) in the absence of 2AAR. Antinociceptive synergy with deltorphin II was preserved in the absence of either 2AAR or 2CAR. In conclusion, ST91 activates both 2AAR and non-2AAR subtypes to produce spinal antinociception and opioid synergy. This study confirms that the spinal pharmacology of ST91 differs from that of other 2AR agonists and extends those data to include spinal synergy with opioid agonists. The unique profile of ST91 may be advantageous in pain management.
2-adrenergic receptor (2AR) family of G-protein-coupled receptors produce antinociception (Kaalzow, 1974
) and synergize with opioid receptor agonists when coadministered in the spinal cord (for review, see Alguacil and Morales, 2004). Adrenergic-opioid synergy is important in pain management because synergy-enabled decreases in dose may improve efficacy and/or reduce side effects (for review, see Walker et al., 2002).
The 2ARs are divided into three distinct subtypes, 2AAR, 2BAR, and 2CAR (Bylund et al., 1994). Identification of distinct physiological roles for the 2AR subtypes may allow for separation of antinociception from sedative and cardiovascular effects. Using genetically modified mice (Link et al., 1995; MacMillan et al., 1996), we and others have previously shown that most 2AR agonists (clonidine, dexmedetomidine, norepinephrine, and UK-14,304) require 2AAR activation to produce antinociception and to synergize with opioid agonists (Hunter et al., 1997; Lakhlani et al., 1997; Stone et al., 1997). An exception is the mixed adrenergic/imidazoline agonist moxonidine, which involves both the 2AAR and 2CAR subtypes in spinal antinociception (Fairbanks et al., 2002). The side effect of sedation, which limits the clinical utility of these compounds, also appears to be mediated by 2AAR activation (Hunter et al., 1997; Lakhlani et al., 1997). The identification and characterization of non-2AAR agonists with antinociceptive but reduced sedative effects is therefore of interest in pain management.
The clonidine analog ST91 has an in vivo pharmacological profile unique among 2AR agonists (Hoefke et al., 1975). For example, i.t. ST91, but not dexmedetomidine or clonidine, is sensitive to antagonism by the non-2AAR-preferring -adrenergic antagonists prazosin, imiloxan, and ARC 239 (Takano and Yaksh, 1992; Takano et al., 1992; Duflo et al., 2002). Furthermore, ST91 does not develop cross-tolerance with dexmedetomidine (Takano and Yaksh, 1993) but rather synergizes with it (Graham et al., 2000), indicating that they probably act upon independent sites. Consistent with this hypothesis, unlike clonidine or dexmedetomidine, ST91 produces minimal or no hypotension or sedation following spinal or systemic delivery (Hoefke et al., 1975; Yasuoka and Yaksh, 1983; Nagasaka and Yaksh, 1990; Dowlatshahi and Yaksh, 1997; Hunter et al., 1997). These and other studies have suggested that antinociception can be mediated by activation of either 2BAR or 2CAR subtypes. Further examination of a potential role for non-2AAR subtypes, however, has been limited by the lack of highly subtype-selective 2AR agonists or antagonists.
In the current study, we used 1) mice with dysfunctional 2AARs due to a targeted point mutation [2AAR (MacMillan et al., 1996)], 2) mice in which the 2CAR gene has been deleted [2CAR-KO (Link et al., 1995)], and 3) pharmacological strategies to characterize the spinal pharmacology of ST91-mediated antinociception and antinociceptive synergy with opioids in the mouse spinal cord. Based on the prior literature, we hypothesized that ST91 would retain potency and synergistic antinociception with opioids in the 2AAR mutant mice but would demonstrate reduced potency and no synergism with opioids in the 2CAR knockout (2CAR-KO) mice. Our findings indicate, however, that ST91 is unique among 2AR agonists in that it activates both 2AAR and non-2AARs to produce spinal antinociception and opioid synergy but has an absolute requirement for neither. ST91 may therefore be of interest in the field of pain management.
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Materials and Methods |
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2AAR-D79N mutant mice were generated by hit-and-run gene targeting as described previously (MacMillan et al., 1996) on a hybrid C57BL/6 and 129/Sv background. Wild-type animals of the same mixed background were used as controls (2AAR-WT). The 2CAR-KO mice were developed in the laboratory of Dr. Brian Kobilka at Stanford University (Link et al., 1995) and purchased from Jackson Laboratory (Bar Harbor, ME) following 17 generations of backcrossing to the C57BL/6 background. C57BL/6 mice pair-bred within our facility were used as wild-type controls (2CAR-WT). Breeding pairs were established, and pups were weaned between 2 and 3 weeks of age. Within each experiment, animals were age- and gender-matched across groups. Animals were used no more than twice. In each case, a rest period of at least 1 week was used, and the animals were randomized across treatment groups. We chose to use these mouse lines because we have extensively characterized their spinal neuropharmacology (Stone et al., 1997; Fairbanks and Wilcox, 1999; Fairbanks et al., 2002), and they have been widely used by other groups with interest in 2AR-mediated antinociception and antihypertensive effects (for review, see Kable et al., 2000). Therefore, the results presented in this study are directly comparable with the prior literature. All experiments were approved by the Institutional Animal Care and Use Committee of the University of Minnesota.
Drug Preparation and Administration. All drugs were dissolved in 0.9% saline. ST91 was a gift of Boehringer Ingelheim Pharmaceuticals USA (Ridgefield, CT). SKF-86466 was a gift of SmithKline & French Laboratories (Philadelphia, PA). BRL-44408 (Tocris, Ellisville, MO), deltorphin II, substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2), prazosin, and idazoxan were all from Sigma (St. Louis, MO). Intrathecal drug administration was done by direct lumbar puncture in a volume of 5 µl according to the method of Hylden and Wilcox (1980) in conscious mice.
Substance P Behavioral Assay. A constant dose (10 ng) of substance P (SP) was administered i.t. in a volume of 5 µl, and the number of caudally directed biting, licking, and scratching behaviors were counted for 1 min following the injection as described previously (Hylden and Wilcox, 1981). For each experimental day, a new control count was obtained, and percent inhibition was determined relative to that control. Control counts typically ranged from between 30 to 40 behaviors per minute. A minimum of six mice were used for each dose. To assess the effects of i.t. administered adrenergic ligands, drugs were coadministered with SP. Results were expressed as the percent inhibition of SP-induced behaviors in comparison with the control group (SP only) according to eq. 1:
 | (1) |
In some experiments, antagonists were coadministered with ST91 and SP. In the case of SKF-86466 and BRL-44408, dose-antagonism curves were determined for their respective abilities to antagonize the inhibition of SP behavior by an approximately 80% effective dose of ST91 (determined empirically each experimental day). The ID80 value is calculated from the regression line of the dose-inhibition curve according to eq. 2:
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Dose-Response Analysis. Individual dose and/or time points are expressed as means with S.E.M. ED50 values and confidence limits were calculated according to the graded dose-response method of Tallarida and Murray (1987) on the linear portion of each dose-response curve. Statistical comparisons of potencies are based on the confidence limits of the ED50 values. A minimum of three doses were used for each drug or combination of drugs. A minimum of 50% was set for a drug to be classified as efficacious.
Isobolographic Analysis. Dose-response curves were constructed for each agonist administered alone, the ED50 values were determined and used to determine an equal potency ratio between the agonists. This ratio was then maintained when both agonists were administered in combination, a third dose-response curve was constructed, and an experimentally derived combination ED50 was calculated. To test for interactions between agonists, the ED50 values and S.E. of all dose-response curves were arithmetically arranged around the ED50 value using eq. 3 (Tallarida, 1992):
 | (3) |
Isobolographic analysis [the appropriate method for evaluating synergistic interactions (Tallarida and Murray, 1987; Tallarida, 1992)] necessitates this manipulation. When testing an interaction between two drugs, a theoretical additive ED50 value is calculated for the combination based on the dose-response curves of each drug administered separately. This theoretical value is then compared by a Student's t test with the observed experimental ED50 value of the combination. These values are based on the total dose of both drugs. An interaction is considered synergistic if the experimental ED50 is significantly less (p < 0.05) than the calculated theoretical additive ED50.
Visualization of drug interactions can be facilitated and enhanced by graphical representation of isobolographic analysis (Figs. 3, B and D, and 4, B and D). This representation depicts the ED50 of each agent as the x-or y-intercept. For example, Fig. 3B presents the ED50 of ST91 as the y-intercept and the ED50 of deltorphin II as the x-intercept in 2AAR-WT. The line connecting these two points depicts the dose combinations expected to yield 50% efficacy if the interaction is purely additive and is called the theoretical additive line. The theoretical additive ED50 and its confidence interval are determined mathematically and plotted spanning this line. The observed ED50 for the combination is plotted at the corresponding x, y coordinates along with its 95% confidence interval for comparison with the theoretical additive ED50. The variability surrounding the observed and theoretical ED50 values represents the vector sum of the vertical and horizontal components of the S.E. to facilitate graphical comparisons between theoretical additive and observed points. All dose-response and isobolographic analyses were performed with the FlashCalc 4.5.3 pharmacological statistics software package generously supplied by Dr. Michael Ossipov.
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Results |
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2AAR-D79N and 2CAR-KO Mice. To determine the relative importance of 2AAR and 2CAR activation in ST91-mediated spinal antinociception, we evaluated the ability of ST91 to inhibit SP-evoked behavior in 2AAR-D79N and 2CAR-KO mice and their WT counterparts. ST91's dose-dependent inhibition of SP-induced behavior was significantly (
3-fold) less potent in 2AAR-D79N compared with 2AAR-WT mice (Fig. 1A). In contrast, ST91 inhibited SP-induced behavior in 2CAR-WT and 2CAR-KO mice with similar potency (Fig. 1B). These data suggest that activation of 2AARs contributes to ST91-mediated spinal antinociception.
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-opioid receptor agonist, deltorphin II was also evaluated in the 2AAR-D79N and 2CAR-KO mice and their respective WT controls. Although deltorphin II was not affected by the loss of functional 2AARs (Fig. 1C), 2CAR deletion resulted in a significant decrease in deltorphin II potency (12-fold), suggesting a role for the 2CAR in deltorphin II-mediated spinal antinociception (Fig. 1D).
Antagonism of ST91 by SKF-86466 (2AR>I1), BRL-44408 (2AAR>2B/CAR), Prazosin (2B/CAR>2AAR), and Pindolol (5-HT1A/1B). The data in Fig. 1, A and B, suggest that neither 2AAR nor 2CAR is required for ST91-mediated antinociception, yet previous studies in rats indicate that the antinociceptive effects of ST91 are mediated by 2ARs. To confirm the involvement of the 2AR family of receptors in ST91-mediated antinociception in mice, we used the nonsubtype-selective, pan-2AR antagonist SKF-86466 (Hieble et al., 1986). SKF-86466 has been shown to have 1000-fold selectivity for 2AR over I1-imidazoline receptors (Yu and Frishman, 1996) and can therefore be used to determine whether a ligand has activity at putative imidazoline receptors. We observed that ST91-mediated antinociception (0.3 nmol i.t.) was antagonized by SKF-86466 in a dose-dependent manner in both 2AAR-WT and 2AAR-D79N mice with similar potency (Fig. 2A). The dose-dependent antagonism of ST91 by SKF-86466 was not altered in 2CAR-KO mice compared with their WT controls (Fig. 2B). These data indicate that ST91-induced antinociception is mediated by 2-adrenergic receptors.
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To further characterize the role of the 2AR subtypes in ST91-mediated antinociception, we evaluated the effect of the 2AAR-preferring antagonist BRL-44408 (Young et al., 1989), which has between 15- and 65-fold higher affinity for 2AAR over 2B/C (Wild et al., 1994; Wikberg-Matsson et al., 1995). ST91 was dose-dependently antagonized by BRL-44408 in both 2AAR-WT and 2AAR-D79N mice (Fig. 2C) but with reduced potency in the 2AAR-D79N mice (115-fold) compared with the 2AAR-WT mice. Because previous data had suggested that moxonidine-induced antinociception is largely independent of 2AAR, we tested BRL-44408 against moxonidine-induced antinociception. BRL-44408 antagonized moxonidine with similar potency in 2AAR-WT and 2AAR-D79N (Fig. 2D), consistent with a non-2AAR site of action for moxonidine. Thus, the decreased antagonistic potency of BRL-44408 against ST91 in 2AAR-D79N mice is unlikely to be due to a nonspecific effect of the 2AAR-D79N mutation, to compensatory mechanisms resulting from the mutation or to strain-related differences. These data are consistent with the hypothesis that 2AARs contribute to but are not necessary for ST91-mediated antinociception. Furthermore, the ability of BRL-44408 to antagonize ST91 confirms that ST91-mediated antinociception requires 2-adrenergic receptor activation.
The non-2AAR-preferring antagonist prazosin has approximately 10- and 25-fold greater affinity at 2BAR and 2CAR, respectively (Wild et al., 1994; Jasper et al., 1998). In 2AAR-WT mice, the addition of prazosin has no effect on ST91-induced antinociception (Fig. 2E). However, ST91-mediated inhibition was sensitive to prazosin antagonism in the 2AAR-D79N mice (Fig. 2E). This observation suggests that a shift from a 2AAR to a non-2AAR site of action occurs in the absence of functional 2AARs. When the 2AAR-requiring agonist dexmedetomidine was tested with prazosin, the antagonist had no effect in either 2AAR-WT or 2AAR-D79N mice (Fig. 2F).
Many 2AR ligands have reported affinities at 5-HT1A subtypes in the nanomolar range (Meana et al., 1996; Newman-Tancredi et al., 1998), including BRL-44408 and SKF-86466. Therefore, antagonism by these ligands does not exclude the possibility that ST91 may be mediating its effects, in part, at 5-HT1A receptors. We therefore tested the ability of the 5-HT1A/1B antagonist pindolol (20 nmol i.t.) to attenuate ST91 and found no effect (Fig. 2G). This dose is in excess of that previously shown to block 5-HT agonist-mediated spinal efficacy (Alhaider and Wilcox, 1993). The mixed 5-HT1/2/7 antagonist methylsergide was similarly ineffective (data not shown). Therefore, ST91-mediated spinal inhibition does not appear to involve an action at 5-HT receptors.
Effects of i.t. Coadministration of ST91 and Deltorphin II. When agonists to both 2AR and opioid receptors are coadministered i.t., they act synergistically to inhibit SP-elicited behavior. We have previously shown that the synergistic interaction between deltorphin II and the 2AR agonist UK-14,304 is 2AAR-dependent (Stone et al., 1997), whereas deltorphin II-moxonidine synergy requires 2CAR activation (Fairbanks et al., 2002). We therefore tested the role of both receptors in deltorphin II-ST91 spinal synergy.
Dose-response analysis in 2AAR-WT mice showed that ST91 was 6-fold more potent than deltorphin II to inhibit SP-induced behavior (Fig. 3A; Table 1). This relative potency fixed the dose ratio for the combination of the two agonists at 1:6 for ST91/deltorphin. The resultant dose-response curve is represented graphically in Fig. 3A in terms of each agonist in the presence of a fixed ratio of the other for ease of visualization. Comparison of the dose-response curves in Fig. 3A demonstrates that the potency of ST91 increased approximately 100-fold in the presence of deltorphin II, suggesting that the interaction is synergistic. If the interaction were additive, the combined presence of an equieffective dose of deltorphin II would have been expected to increase ST91 potency by roughly 2-fold. The dose-response data from Fig. 3A are represented graphically as an isobologram in Fig. 3B. As shown in the graph, the actual total combined ED50 for 2AAR-WT mice (Fig. 3B, closed circle) is lower than the theoretical additive ED50 (Fig. 3B, open circle), indicating that this interaction is synergistic. This synergistic interaction was confirmed by statistical comparison (Student's t test) between the observed combined ED50 value and the theoretical additive ED50 value (Table 1).
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The corresponding data for 2AAR-D79N mice are depicted in Fig. 3C. Intrathecally administered ST91, deltorphin II, or both combined inhibited SP-elicited behavior in a dose-dependent manner (Fig. 3; Table 1). The ST91/deltorphin II potency ratio of 1:1 determined the dose ratio for coadministration. Comparison of the dose-response curves for ST91 in the presence (open squares) and absence (closed squares) of deltorphin in Fig. 3C demonstrates that the potency of ST91 increased approximately 100-fold in the presence of deltorphin II, suggesting a synergistic interaction. This result is further illustrated by the nonoverlapping S.E.s between observed and theoretical ED50 values in Fig. 3D, indicating that this interaction is synergistic in 2AAR-D79N mice. This synergistic interaction was confirmed by statistical comparison (t test) of the observed combined ED50 value with the theoretical additive ED50 value (Table 1). These results document that ST91/deltorphin II spinal synergy does not require functional 2AARs.
We have demonstrated previously that the ability of the 2AR agonist moxonidine to synergize with deltorphin II following i.t. delivery was dependent on 2CAR, not 2AAR activation. We therefore examined the interaction between ST91 and deltorphin II in 2CAR-WT and 2CAR-KO mice. As shown in Fig. 4, the synergistic interaction observed in 2CAR-WT mice (Fig. 4, A and B; Table 1) is retained in the absence of the 2CAR (Fig. 4, C and D; Table 1). These synergistic interactions were confirmed by statistical comparison (Student's t test) between the observed combined ED50 value and the theoretical additive ED50 value (Table 1). These data suggest that ST91/deltorphin II spinal synergy requires exclusive activation of neither 2AAR nor 2CAR.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionConclusionsReferences |
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2ARs (three subtypes, 2AAR, 2BAR, and 2CAR) produce antinociception and synergize with opioids. Most 2AR agonists require 2AAR activation to produce spinal antinociception and opioid synergy. The fact that the 2AAR also mediates the side effect of sedation limits the clinical utility of these compounds. Previous studies have suggested that the 2AR agonist ST91 has a nonsedating, non-2AAR mechanism of action.
The present study examined the spinal pharmacology of ST91 and its interaction with the -opioid agonist deltorphin II in mice lacking either functional 2AARs or 2CARs. Our data indicate that ST91 can produce spinal 2AR-mediated antinociception and opioid synergy in the absence of either the 2AAR or the 2CAR subtype. It is possible that undefined developmental and/or compensatory changes could account for the retention of ST91-mediated potency and opioid synergism the 2AAR or the 2CAR mutant mice, perhaps by up-regulation of other 2ARs. However, the loss of function of other 2AR agonists previously reported in these animals suggests this in unlikely (Stone et al., 1997; Fairbanks et al., 2002). This unique profile of ST91 may be advantageous in pain management.
Potential Antinociceptive Target(s) of ST91. The apparently conflicting observations regarding the role(s) of the 2AAR, 2BAR, and 2CAR subtypes in spinal adrenergic antinociception and/or antihyperalgesia can be largely explained on the basis of ligand selectivity. Pharmacological studies suggesting that clonidine-induced spinal antinociception is mediated by the 2AAR subtype (Takano and Yaksh, 1992, 1993; Takano et al., 1992; Duflo et al., 2002) are consistent with the loss of spinal clonidine efficacy in 2AAR-D79N mice (Fairbanks and Wilcox, 1999). Similar agreement between pharmacological and genetic studies exists for dexmedetomidine (Takano and Yaksh, 1992; Hunter et al., 1997; Lakhlani et al., 1997; Stone et al., 1997).
Pharmacological studies have consistently demonstrated that ST91 acts at a site distinct from that of clonidine or dexmedetomidine based on the following observations: 1) lack of cross-tolerance between ST91 and dexmedetomidine (Takano and Yaksh, 1993), 2) differential sensitivity of dexmedetomidine and clonidine versus ST91 to antagonism by atipamezole and idazoxan (Takano and Yaksh, 1992; Takano et al., 1992); 3) sensitivity of ST91 but not dexmedetomidine or clonidine to antagonism by prazosin and imiloxan (Takano and Yaksh, 1992; Takano et al., 1992), 4) the existence of antinociceptive synergy between ST91 and dexmedetomidine (Graham et al., 2000), and 5) antagonism of clonidine but not ST91 by BRL-44408 (Duflo et al., 2002). The current finding that ST91-mediated spinal antinociception is only slightly attenuated in the absence of functional 2AARs [as opposed to nearly complete abolition of dexmedetomidine and clonidine-mediated spinal antinociception (Stone et al., 1997; Fairbanks and Wilcox, 1999)] is consistent with the aforementioned pharmacological studies.
The current data raise the question as to whether ST91 antinociception is mediated by the 2BAR, the 2CAR, or non-2AR sites. The present study reports that ST91-mediated antinociception is normal in 2CAR-KO mice, ruling out that receptor as the sole site of action. However, the dose-dependent antagonism of ST91 by the pan-2AR antagonist, SKF-86466, also suggests that I1-imidazoline receptor binding sites are not involved (SKF-86466 has >1000-fold affinity at 2ARs versus the putative I1-imidazoline receptor). Although it is not known if ST91 binds to 5-HT1A receptors, both SKF-86466 and BRL-44408 have affinities in the high nanomolar range at this receptor subtype (Meana et al., 1996). It is therefore possible that 5-HT1A receptors may be targeted by ST91. To rule out this possibility, we tested the ability of the 5-HT receptor antagonists methylsergide and pindolol (Alhaider and Wilcox, 1993) to antagonize ST91-mediated spinal inhibition. ST91 was insensitive to these agents (Fig. 2), suggesting that the most likely site of antinociceptive action for ST91 is at 2ARs.
The current data do not distinguish between the possibilities that 1) ST91 adopts the 2CAR as its primary target in the absence of 2AAR, or 2) ST91 produces antinociception by acting at 2BARs. The potential involvement of 2BAR in spinal adrenergic antinociception has been previously suggested. Perhaps the strongest evidence comes from Maze and colleagues (Sawamura et al., 2000), who have shown that the antinociceptive effect of nitrous oxide (N2O) is significantly attenuated in 2BAR-KO mice. The role of the 2BAR in the central nervous system (CNS) is underexplored compared with the other subtypes. This is due, in part, to earlier reports that 2BAR mRNA expression in the CNS is limited to low levels in the thalamus (Nicholas et al., 1993; Scheinin et al., 1994). However, as methodologies improved, 2BAR mRNA was detected in other CNS regions [i.e., dorsal root ganglia (Gold et al., 1997; Shi et al., 2000)]. Identification of a role for the 2BAR in spinal antinociception is therefore not inconsistent with its localization. Although further studies are needed, the 2BAR may play an important, but largely unexplored, role in antinociception.
Data from in vitro binding studies suggest that most 2AR agonists have similar affinity across the 2AR subtypes; for example, ST91 has equivalent pKis(2AAR = 6.24, 2BAR = 6.10, 2CAR = 6.21); clonidine also has similar pKis (2AAR = 7.21, 2BAR = 7.16, 2CAR = 6.87) (Jasper et al., 1998). The lack of agonist selectivity across receptor subtype raises the question why clonidine- but not ST91-mediated spinal antinociception is completely abolished in the absence of functional 2AARs in vivo. One possible explanation is related to pharmacodynamic properties. Although clonidine is highly lipophilic (octanol/buffer partition coefficient of 3.2), ST91 is highly hydrophilic (partition coefficient of 0.05) (Kobinger and Pichler, 1975). Poor lipid solubility may limit ST91 access to the CNS as opposed to ST91 lacking affinity at central 2AARs; limited access may explain why ST91 does not produce sedation or hypotension after systemic administration (Hoefke et al., 1975). Likewise, poor penetration deep into the spinal cord may explain the reported lack of sedation and hypotension following i.t. injection of ST91.
ST91 and Opioid Antinociceptive Synergy. The ability of 2AR agonists to synergize with opioids is an important aspect of their potential as therapeutic agents. In cases where synergy exists, the doses of each drug required to reach a given level of effect are significantly decreased in the presence of the other compound. Synergy-enabled decreases in dose, in turn, present an opportunity to simultaneously reduce side effects and maximize efficacy (for comprehensive review, see Alguacil and Morales, 2004). ST91 had been shown previously to synergize with morphine following spinal delivery (Monasky et al., 1990). The current study reports a synergistic interaction between ST91 and the -opioid receptor agonist deltorphin II when both agents are coadministered spinally. Unlike the synergistic interaction between deltorphin II and the 2AR agonists UK-14,304 and moxonidine, which require the 2AAR or the 2CAR, respectively, the synergistic interaction between ST91 and deltorphin II is preserved in the absence of either 2AAR or 2CAR. This finding underscores the unique pharmacology of ST91 and suggests that either 1) ST91 can synergize with deltorphin II through an action at 2BAR or that 2) activation of either 2AAR or 2CAR is sufficient to produce synergy.
In this study, we observed a significant decrease in deltorphin II potency in the 2CAR-KO mice. Descending noradrenergic input to the spinal cord contributes to spinal opioid antinociception. The loss of an otherwise synergistic interaction between deltorphin II and endogenous NE targeting 2CAR would explain this observation. This observation suggests that a component of descending noradrenergic tone may involve activity at 2CAR. However, further studies are needed to determine 1) whether this phenomenon generalizes to other opioid agonists and 2) the relative contributions of the 2AR subtypes in descending noradrenergic antinociception.
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Conclusions |
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TopAbstractMaterials and MethodsResultsDiscussionConclusionsReferences |
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2AAR or 2CAR but remains -2 adrenergic in nature. These data suggest that either the 2BAR contributes to ST91's effects in the mouse spinal cord or that ST91 has similar potency at both 2AAR and 2CAR and can produce antinociception at either of these receptors in the absence of the other. Further studies are needed to distinguish between these possibilities. Development of agonists that utilize other 2AR subtypes in addition to or in lieu of 2AARs may represent an improvement over current therapies directed at the 2AAR subtype, which also mediates 2AR agonist-induced hypotension and sedation (MacMillan et al., 1996; Lakhlani et al., 1997). It may therefore be possible to improve the therapeutic window of 2AR agonists in pain management by combination treatment to more fully take advantage of their powerful antinociceptive and antihyperalgesic actions. Finally, the synergistic interaction between ST91 and the opioids morphine (Monasky et al., 1990) and deltorphin II indicate the utility of ST91 as an opioi-denhancing adjunct therapy. |
Acknowledgements |
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2AAR-D79N mice and wild-type counterparts. |
Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: 2AR, 2-adrenergic receptor; UK-14,304, 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine; prazosin, 4-(4-amino-6,7-dimethoxy-quinazolin-2-yl) piperazin-1-yl]-(2-furyl)methanone; ARC 239, 2-(2,4-(O-methoxyphenyl)-piperazin-1-yl)ethyl-4,4-dimethyl-1, 3-(2H,4H)-isoquinolindione; KO, knockout; WT, wild type; ST91, 2-(2,6-diethylphenylamino)-2-imidazoline hydrochloride; SKF-86466, 6-chloro-2,3,4,5-tetrahydro-3-methyl-1-H-3-benzazepine; BRL-44408, 2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidazole maleate; deltorphin II, Tyr-D-Ala-Phe-Glu-Val-Val-Gly amide; idazoxan, (±)-2-[1,4-benzodioxan-2-yl]-2-imidazoline hydrochloride; SP, substance P, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2; 5-HT, 5-hydroxytryptamine; CNS, central nervous system; CI, confidence interval.
Address correspondence to: Laura S. Stone, Faculty of Dentistry, McGill Centre for Research on Pain, 3640 University Street, Montreal, Quebec H3A 2B2, Canada. E-mail: laura.s.stone{at}mcgill.ca
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) and deltorphin II (
) inhibited the behavior in a dose-dependent manner. The agonists were then coadministered at a constant ST91/deltorphin dose ratio of 1:6 (
, ST91; 
deltorphin). This dose ratio was based on the relative potencies of ST91 and deltorphin (5.4:1) administered alone. B, isobolographic analysis applied to the data from Fig. 3A. The y-intercept represents the ED50 for ST91, and the x-intercept represents the ED50 for deltorphin II. The lines directed from each ED50 value toward zero represent the respective lower 95% confidence limits of each ED50. The line connecting these two points is the theoretical additive line. The open circle on the theoretical additive line represents the calculated theoretical ED50 value of the combination if the interaction is additive. The line segments through the two ED50 points and directed along the line joining them represents the vector sum of the vertical and horizontal S.E. of each ED50. The observed combination ED50 (
