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TOXICOLOGY

Mechanisms Responsible for the Enhanced Antinociceptive Effects of µ-Opioid Receptor Agonists in the Rostral Ventromedial Medulla of Male Rats with Persistent Inflammatory Pain

Departments of Anesthesia (S.R.W., D.L.H.) and Pharmacology (K.T.S., D.L.H.) and the Medical Scientist Training Program (K.T.S.), The University of Iowa, Iowa City, Iowa; Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin (H.M., L.F.T.); and Department of Anesthesiology and Critical Care, The Johns Hopkins University, Baltimore, Maryland (R.W.H.)

Received February 25, 2007; accepted May 8, 2007.

	Abstract

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This study investigated three possible mechanisms by which the antinociceptive effects of the µ-opioid receptor (MOR) agonist [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (DAMGO) and the -opioid receptor (DOR) agonist [D-Ala²,Glu⁴]-deltorphin (deltorphin II) (DELT), microinjected into the rostral ventromedial medulla (RVM), are enhanced in rats with persistent inflammatory injury. Radioligand binding determined that neither the B_max nor the K_d values of [³H]DAMGO differed in RVM membranes from rats that received an intraplantar injection of saline or complete Freund's adjuvant (CFA) in one hindpaw 4 h, 4 days, or 2 weeks earlier. Likewise, neither the EC₅₀ nor the E_max value for DAMGO-induced stimulation of guanosine 5'-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPS) binding differed in the RVM of saline- or CFA-treated rats at any time point. Microinjection of fixed dose combinations of DAMGO and DELT in the RVM of naive rats indicated that these agonists interact synergistically to produce antinociception when DAMGO is present in equal or greater amounts than DELT and, additively, when DELT is the predominant component. Thus, unlike the periphery or spinal cord, potentiation of MOR-mediated antinociception does not entail an increase in MOR number, affinity, or coupling. Rather, the data are concordant with our proposal that potentiation results from a synergistic interaction of exogenous MOR agonist with DOR-preferring enkephalins whose levels are increased in CFA-treated rats (J Neurosci 21:2536–2545, 2001). Virtually no specific [³H]DELT binding nor stimulation of [³⁵S]GTPS binding by DELT was obtained in RVM membranes from CFA- or saline-treated rats at any time point. The mechanisms responsible for the potentiation of DELT-mediated antinociception remain to be elucidated.

Levels of MOR and DOR mRNA and protein are also increased in the ipsilateral dorsal horn of the spinal cord under conditions of inflammatory injury (Ji et al., 1995; Maekawa et al., 1995; Goff et al., 1998; Cahill et al., 2003). Although this increase may reflect up-regulation of MOR and DOR in the central terminals of primary afferent neurons, increased trafficking of DOR to the plasma membrane of neurons in the dorsal horn also occurs in CFA-treated rodents (for review, see Cahill et al., 2007). Although visceral inflammation similarly increases the levels of DOR mRNA in the dorsal horn, levels of DOR protein are not significantly increased (Pol et al., 2003).

The antinociceptive effects of the MOR agonist [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (DAMGO) and the DOR agonist [D-Ala²,Glu⁴]-deltorphin (deltorphin II) (DELT) microinjected into the rostral ventromedial medulla (RVM) are also enhanced in rats with persistent inflammatory injury (Hurley and Hammond, 2000). However, little is known about the mechanisms by which this occurs. The present study investigated three possibilities. The first set of experiments used radioligand binding to determine whether the number or affinity of MOR or DOR in the RVM increases 4 h, 4 days, or 2 weeks after intraplantar injection of CFA in one hindpaw. These time points were chosen because the antinociceptive effects of DAMGO and DELT are enhanced in a time-dependent manner with maximal enhancement evident 2 weeks after CFA injection. The second set of experiments examined GTPS binding to determine whether MOR or DOR couple to G_i/o with greater efficiency 4 h, 4 days, or 2 weeks after injection of CFA. Finally, prior work demonstrated that the enhanced antinociceptive effect of DAMGO is reversed by coadministration of a DOR antagonist and that levels of [Met⁵]-enkephalin and [Leu⁵]-enkephalin increase in the RVM of rats with persistent inflammatory injury (Hurley and Hammond, 2001). These findings led us to postulate that the enhancement of the antinociceptive effect of DAMGO was the result of an additive or synergistic interaction between the exogenous MOR agonist and the endogenous enkephalins, which have preferential affinity for DOR (Hurley and Hammond, 2001). Although a logical extension of a large literature documenting a synergistic interaction of MOR and DOR agonists in the central nervous system (Malmberg and Yaksh, 1992; Adams et al., 1993), this proposal has not been directly tested in the RVM. Therefore, the final set of experiments used a fixed dose ratio analysis to determine whether DAMGO and DELT interact in an additive or synergistic manner to produce antinociception after concurrent microinjection in the RVM of naive rats.

	Materials and Methods

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Adult male Sprague-Dawley rats (Charles River, Raleigh, NC) were used in these studies. The experiments were approved by the University of Iowa Animal Care and Use Committee, and they were conducted in accordance with the Guide for Care and Use of Laboratory Animals as promulgated by the National Institutes of Health and the guidelines of the International Association for the Study of Pain. Every effort was made to minimize the number of rats used and their suffering.

Assessment of Nociceptive Threshold and Paw Thickness.

Rats were acclimated to the testing environment for 30 min and then placed in individual Plexiglas chambers situated on a glass surface that was maintained at 25°C for another 15-min period of acclimation. Nociceptive sensitivity was measured using the paw withdrawal test in which a high-intensity beam of light was focused on the plantar surface of the hindpaw. The time required for the rat to remove its hindpaw from the thermal stimulus was termed the paw withdrawal latency (PWL). The high-intensity lamp was adjusted to elicit baseline paw withdrawal latencies of 8 to 12 s. If a withdrawal response did not occur within 20 s, the test was terminated to prevent tissue injury, and the rat was assigned this latency. Response latencies were determined for the left and right hindpaw and then averaged. Hindpaw thickness in the dorsoventral axis was measured with digital calipers.

Opioid Receptor Binding. After baseline PWL and hindpaw thickness were determined, rats were lightly anesthetized with halothane and injected with 150 µl of CFA (150 µgof Mycobacterium butyricum, 85% Marcol 52, and 15% Aracel A mannide monoemulsifier; Calbiochem, San Diego, CA) or saline, pH 7.4, in the left hindpaw. Rats were returned to their cages for 4 h, 4 days, or 2 weeks. These experiments were conducted using cohorts of rats in which rats from each shipment (18–24 at a time) were allocated among the six different treatment conditions (i.e., CFA or saline injection; 4-h, 4-day, or 2-week survival time). Multiple replicates of each cohort were performed.

On the designated day, PWL and paw thickness were redetermined to verify the presence of thermal hyperalgesia and inflammation in CFA-treated rats. Animals were then euthanized by CO₂ inhalation. A 3-mm-thick transverse section through the medulla was rapidly dissected out and placed on an ice-cold Petri dish. A triangular region was dissected free, and it was immediately frozen on dry ice. The apex of the triangle was on the midline at the ventral edge of the ventricle. The two remaining corners were situated at the lateral edges of the dorsal aspect of each pyramid. This region contained the RVM, which included the nucleus raphe magnus (NRM) and the adjacent, bilateral nucleus reticularis gigantocellularis pars alpha (NGCp). Tissue was stored at –70°C.

Tissue containing the RVM from three to four rats in each treatment group was pooled and homogenized in 20 volumes (w/v) of membrane buffer (50 mM Tris, pH 7.4) using Potter-Elvehjem homogenizer tubes and Teflon pestles. After centrifugation at 39,000g for 15 min at 4°C, the pellet was resuspended in buffer, homogenized, and allowed to incubate for 10 min at 25°C to allow for dissociation and degradation of endogenous peptides. The suspension was centrifuged again at 39,000g for 15 min at 4°C. The resultant pellet was resuspended in buffer, homogenized, and stored at –70°C. Protein content was determined by the Lowry method using bovine serum albumin as the standard (Lowry et al., 1951). Membrane preparations were coded to blind the investigator to treatment condition.

Preliminary association and dissociation analyses were conducted to establish optimal binding conditions, and linearity of binding was confirmed between 25 and 200 µg of protein/tube. The saturation binding assay was conducted in triplicate. Membranes (100–120 µg/tube) were incubated in increasing concentrations of [³H]DAMGO (0.03–10 nM) for 1 h or [³H]DELT (0.03–6 nM) for 2 h, in the presence and absence of naloxone at 25°C. The reaction was terminated by rapid filtration through Whatman GF/B glass fiber filters (Whatman, Clifton, NJ) in a cell harvester (Brandel Inc., Gaithersburg, MD) and three 5-ml washes with ice-cold buffer (50 mM Tris, pH 7.2). The filter paper was soaked in buffer solution containing 50 mM Tris, pH 7.2, and 0.01% polyethylenimine for at least 2 h before membrane harvesting to reduce nonspecific binding. Filters were equilibrated overnight in scintillation counting fluid and counted in a liquid scintillation analyzer (LS 6500; Beckman Coulter, Fullerton, CA).

Radioligand binding data are reported as the mean ± S.E.M. values of three to four experiments, each of which contained RVM tissue from three to four rats. Specific binding was determined as the difference between total binding in the absence and presence of 1 µM naloxone for MOR and 10 µM naloxone for DOR. Saturation binding curves were fit by nonlinear regression, and an F-test was conducted to determine whether the curves best fit a one- or two-site model. Nonlinear regression methods were used to determine B_max and K_d values for each radioligand using GraphPad Prism version 4 (GraphPad Software Inc., San Diego, CA). The B_max and K_d values determined for each experiment were averaged to generate the mean and S.E.M. Two-way analysis of variance was used to compare B_max and K_d values in saline- and CFA-treated rats at each time point. In this analysis, treatment was one factor and time was the second factor. A p < 0.05 was considered significant.

[³⁵S]GTPS Binding. Animals were prepared as described above for the radioligand binding study. Membrane homogenates containing the RVM were prepared by pooling tissue from three to six rats. Tissue was suspended in membrane buffer (50 mM Tris, pH 7.4, 5 mM MgCl₂, and 1 mM EGTA), homogenized using glass Potter-Elvehjem homogenizer tubes and Teflon pestles, and centrifuged at 39,000g for 15 min at 4°C. The pellet was resuspended in assay buffer (50 mM Tris, pH 7.4, 5 mM MgCl₂, 1 mM EGTA, and 100 mM NaCl), incubated at 25°C for 10 min, and recentrifuged at 39,000g at 4°C. The resultant pellet was suspended in assay buffer and frozen at –70°C for up to 3 months. Protein concentration was determined by the Lowry method. Membrane preparations were coded to ensure that the investigator was blind to treatment condition.

Membranes were thawed and incubated for 10 min at 30°C in the presence of 13.7 mU/ml adenosine deaminase before addition to reaction tubes. Adenosine deaminase was used to decrease residual contributions of adenosine-induced G protein coupling to background levels. [³⁵S]GTPS binding curves were generated by incubating membranes (20–40 µg/tube) for 2 h at 30°C in increasing concentrations of DAMGO (0.001–10 µM) or DELT (0.001–10 µM), in the presence of 30 µM GDP (for DAMGO) or 300 µM GDP (for DELT) and 50 pM [³⁵S]GTPS. Nonspecific binding was determined in the presence of 10 µM GTPS. Samples were run in duplicate. The reaction was terminated by rapid filtration using a cell harvester (Brandel Inc.) fitted with GF/B glass filters (Whatman) presoaked for at least 2 h in buffer solution (5 mM MgCl₂ and 50 mM Tris, pH 7.4, at 4°C). Membranes were washed three times with 5 ml of ice-cold buffer solution (50 mM Tris, pH 7.4). Individual filters containing bound membrane equilibrated overnight in scintillation fluid (ScintiSafe Econo 1; Fisher Scientific, Waltham, MA), and they were counted in a liquid scintillation analyzer (LS 6500; Beckman Coulter).

[³⁵S]GTPS binding data are reported as mean ± S.E.M. of three to four independent replicates, each of which contained RVM tissue from three to six rats. Specific binding was determined by subtracting nonspecific from total [³⁵S]GTPS binding determined in the presence and absence of 10 µM GTPS, respectively. Binding data were expressed as percentage of change from control [(specific binding in the presence of drug–specific binding in the absence of drug)/specific binding in the absence of drug) x 100)]. Specific binding and percentage of [³⁵S]GTPS stimulation were fit with a logistic equation to determine E_max and EC₅₀ values with GraphPad Prism (San Diego, CA). The E_max and EC₅₀ values determined for each experiment were averaged to generate mean and S.E.M. Two-way analysis of variance was used to compare the effects of CFA injection with that of saline on E_max and EC₅₀ values. Treatment (CFA or saline) was one factor, whereas time (4 h, 4 days, or 2 weeks) was the second factor. A p < 0.05 was considered significant.

Microinjection Study. Rats were anesthetized with a mixture of ketamine (70 mg/kg i.p.) and xylazine (11 mg/kg i.p.). Then, they were placed in a stereotaxic instrument and implanted with an intracerebral guide cannula (26-gauge; Plastics One, Roanoke, VA) that terminated 3 mm dorsal to the RVM. Cannulae were affixed to the skull with stainless steel screws and dental acrylic, and a stainless steel stylet was inserted in each guide cannula to maintain patency. After surgery, animals were housed individually and maintained on a 12-h light/dark cycle with free access to food and water. Cannulae were implanted 7 to 10 days before behavioral testing.

Following determination of baseline PWL, DELT (0.03 ng–1.25 µg), DAMGO (0.01–30 ng), or a mixture of DELT and DAMGO in a fixed dose ratio was microinjected into the NRM. For microinjection, drugs were dissolved in saline, or, for the highest dose of DELT, they were dissolved in distilled water. Microinjections into the NRM were made in a volume of 0.25 µl via a 33-gauge stainless steel injector needle that extended 3 mm beyond the guide cannula tip. Drug delivery was monitored by following the movement of an air bubble in the tubing that connected the injector to the syringe pump. The needle was left in place for 60 s after the injection to minimize diffusion of drug up the injection tract. Paw withdrawal latency was redetermined 15, 30, and 60 min later. Each rat received only one dose and was used once. The investigator was blinded to the test drug. Prior work demonstrated that the peak effect of DAMGO is delayed by 15 min when injected in the adjacent NGCp (Hurley et al., 2003). Therefore, the few microinjection sites that were located in the NGCp were excluded from this analysis.

Whether a drug mixture will exhibit additivity or synergy is a function of the dose ratio, because the contribution of each constituent to the observed effect depends on its individual potency and its fractional quantity in the mixture (Tallarida, 2000). These experiments therefore used a fixed dose ratio approach, in which dose-response relationships were constructed for mixtures of DAMGO and DELT where the ratio between each constituent was held constant throughout the total dose range tested. Three different dose ratios were examined. The first dose ratio (1:273; DAMGO:DELT) approximated the ratio of the ED₅₀ values of each drug given alone. Dose ratios of 1:1 and 10:1 (DAMGO:DELT) were also examined because the variance of the theoretical additive dose-response relationship (Z_add) for these dose ratios was minimal (0.15 and 0.18), which facilitated detection of significant differences between the theoretical additive line and the experimentally determined dose-response relationship (Tallarida, 2000).

After testing, animals were euthanized by CO₂ inhalation. Brains were removed and fixed by immersion in 10% formalin containing 30% sucrose. Transverse sections (25 µm in thickness) were cut on a cryostat microtome, and they were stained with cresyl violet to identify the injection site. Injection sites were identified by a person blinded to the behavioral outcome. Sites were plotted on an atlas of the rat brain and then verified by a second individual also blinded to behavioral outcome.

Statistical Analysis. Data are reported as mean ± S.E.M. A two-way analysis of variance for repeated measures was used to compare the effects of DAMGO, DELT, or the drug combination to saline. Dose-response relationships were determined by least-squares linear regression analysis of PWL measured at 15 min, the time of peak effect for the individual drugs and the drug combinations. The ED₅₀ value was defined as the dose that produced 50% of the maximal increase in PWL. The mean baseline paw withdrawal latency was 10 s, and the maximum average response latency was 18 s. Therefore, the criterion latency was set to 14 s for calculation of ED₅₀ values. The 95% confidence limits (CL) were determined as described by Tallarida (2000). For drug mixtures, the dose was expressed as the total dose (DELT + DAMGO) of drug administered in mass units.

The slopes of the dose-response curves for DAMGO and DELT administered alone differed significantly from one another. Moreover, the limited solubility of DELT did not allow us to determine that its maximum efficacy was equivalent to DAMGO. These issues render isobolographic analysis significantly more complex. For example, differences in slope necessitate the construction of two symmetrical isoboles (Tallarida, 2007). In addition, differences in efficacy result in nonlinear isoboles that reach their asymptote at levels of effect greater than the E_max of the least efficacious constituent (Tallarida, 2007). Therefore, isoboles were not constructed for this study. Rather, the experimentally derived dose-response relationship for each mixture was plotted relative to the dose-response relationship for each constituent alone and to the theoretical dose-additive relationship for that particular mixture. Because the highest amount of DELT in the 1:1 and 10:1 mixtures never exceeded 10 ng, a dose that was without effect by itself and was at least 60-fold lower than the minimally effective dose of DELT, it was also possible to calculate the theoretical dose-additive lines for the 1:1 and 10:1 mixtures as if DAMGO had been mixed with an inactive agent and to compare the theoretical and experimentally determined dose-effect relationships using standard "parallel line" assay methods (Tallarida, 2000). In addition, two-tailed t tests were used to compare the experimentally derived ED₅₀ value and its variance to the calculated theoretical dose additive ED₅₀ value and its variance at a predetermined level of effect; in this case, a PWL of 14 s (Tallarida, 2000). A p < 0.05 was considered significant.

Drugs and Reagents. DAMGO, DELT, GDP, NaGTP, naloxone hydrochloride, and adenosine deaminase were purchased from Sigma-Aldrich (St. Louis, MO). [³H]DAMGO (60 Ci/mmol) was purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK). [2-D-Ala],Tyrosyl-3,5-[³H]deltorphin II ([³H]DELT; 45 Ci/mmol) was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). [³⁵S]GTPS (1000 Ci/mmol) was purchased from GE Healthcare. GTPS was purchased from Fisher Scientific

	Results

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Intraplantar Injection of CFA Induces Thermal Hyperalgesia and Hindpaw Inflammation. Table 1 illustrates that ipsilateral PWL was significantly decreased 4 h, 4 days, and 2 weeks after intraplantar injection of CFA compared with saline. Inflammation was also present throughout this period. Response latencies and thickness of the contralateral hindpaw did not differ from those of saline-treated rats.

Persistent Inflammatory Injury Does Not Alter Opioid Receptor Number or Affinity in the RVM. Table 2 illustrates that the B_max values for MOR in RVM membranes from saline- and CFA-treated rats did not differ at any time point (p > 0.7). Likewise, the K_d value of DAMGO did not differ between saline- and CFA-treated animals at any time point (p > 0.3). Although B_max values for DAMGO in the 2-week time point in both CFA- and saline-treated rats seemed to be modestly decreased compared with the 4-h or 4-day treatment groups, this decrease did not achieve statistical significance (p = 0.055 for saline and p = 0.18 for CFA). The basis for this decrease is unknown, but its occurrence in both groups may relate to the longer exposure of these rats to a new housing environment and diet after receipt from the vendor. Figure 1A depicts a representative saturation isotherm for [³H]DAMGO in RVM tissue from saline- and CFA-treated rats. Virtually no specific [³H]DELT binding was detected in membrane homogenates of the RVM from either CFA- or saline-treated rats at any time point (data not shown).

View larger version (13K): [in this window] [in a new window]   Fig. 1. A, representative saturation isotherms for specific binding of [3H]DAMGO to membrane homogenates containing the rostral ventro-medial medulla of rats 2 weeks after intraplantar injection of CFA (open squares) or saline (filled squares) in the hindpaw. Maximal enhancement of DAMGO or DELT-induced antinociception occurs 2 weeks after CFA injection (Hurley and Hammond, 2000). Results from a single experiment, in which tissue from three to four rats was pooled in each treatment group, are illustrated. Nonspecific binding was determined in the presence of 1 µM naloxone. Symbols are the mean ± S.E.M. of triplicate determinations. Ordinate, femtomoles of [3H]DAMGO bound per milligram of protein. Abscissa, concentration of [3H]DAMGO. B, stimulation of [35S]GTPS binding to membrane homogenates containing the rostral ventromedial medulla of rats 2 weeks after intraplantar injection of CFA (open squares) or saline (filled squares) in the hindpaw. Symbols are the mean ± S.E.M. of three different experiments. Nonspecific binding was determined in the presence of 30 µM unlabeled GTPS. Ordinate, percentage of stimulation of basal [35S]GTPS. Abscissa, concentration of DAMGO.

Persistent Inflammatory Injury Does Not Alter Opioid Stimulation of GTPS Binding in the RVM. Table 3 presents the E_max and EC₅₀ values for DAMGO-induced stimulation of [³⁵S]GTPS binding in RVM membranes of saline- and CFA-treated rats. E_max values did not differ between treatment groups at any time point (p > 0.6). Likewise, there was no difference in the EC₅₀ of DAMGO between saline- and CFA-treated animals at any time point (p > 0.3). Figure 1B illustrates the concentration-response curves for DAMGO-induced [³⁵S]GTPS binding to membranes prepared from the RVM of rats 2 weeks after intraplantar injection of CFA or saline. It was not possible to obtain reliable specific binding for DELT-induced [³⁵S]GTPS binding at any time point in RVM membrane homogenates prepared from either saline- or CFA-treated rats. In contrast, a positive control experiment demonstrated that 10 µM DELT stimulated [³⁵S]GTPS binding by 47.2% in homogenates of the striatum.

View this table: [in this window] [in a new window]   TABLE 3 Peripheral inflammatory injury does not alter the potency (EC50) or efficacy (Emax) of DAMGO to stimulate binding of [35S]GTPS in the rostral ventromedial medulla Concentration-response curves were generated for DAMGO-induced stimulation of the binding of [35S]GTPS to membranes containing the rostral ventromedial medulla of rats 4 h, 4 days, or 2 wk after intraplantar injection of saline or CFA in one hindpaw. Values are the mean ± S.E.M. of three to four independent experiments. Data are the percentage of change from basal values in the absence of DAMGO.

MOR and DOR Agonists Can Interact in an Additive or Supra-Additive Manner in the RVM. Microinjection sites were distributed throughout the rostrocaudal extent of the NRM consistent with previous reports from this laboratory (Hurley and Hammond, 2000; Hurley et al., 2003). Figure 2 presents photomicrographs of a microinjection site in the NRM of two different rats. Microinjection of DAMGO, DELT, or the drug combination into sites outside the NRM, such as the nucleus gigantocellularis, the pyramids, and the inferior olive, was either ineffective or only marginally effective, and these sites were excluded from further analysis.

View larger version (124K): [in this window] [in a new window]   Fig. 2. Photomicrographs of a microinjection site (arrow) in the nucleus raphe magnus of two different representative animals (A and B). The coronal sections are stained with cresyl violet. G, genu of the seventh cranial nerve; icp, inferior cerebellar peduncle; P, pyramids, VII, facial motor nucleus.

Microinjection of 0.1 to 30 ng of DAMGO in the NRM increased PWL in a dose- and time-dependent manner. Paw withdrawal latency was maximally increased at 15 min, and then it decreased over the subsequent hour (illustrated for 30 ng in Fig. 3A). Microinjection of 1.25 µg of DELT in the NRM also maximally increased PWL at 15 min with a subsequent decrease over time (Fig. 3A). Doses higher than 1.25 µgof DELT were not soluble. Lower doses of DELT (0.62 µg, 0.31 µg, 3 ng, and 0.3 ng; n = 4–7 per each group) did not increase PWL (p > 0.3). The time course of the increase in PWL produced by each of the fixed dose ratio combinations of DAMGO and DELT was identical to that of the agonists alone. Figure 3B illustrates that the peak increase in PWL produced by the highest dose combination for each fixed dose ratio also occurred at 15 min and that the dose combinations exhibited a time course similar to that of each agonist alone.

View larger version (17K): [in this window] [in a new window]   Fig. 3. A, time course of the increase in PWL produced by microinjection of the highest effective dose of DAMGO (30 ng; n = 7) or DELT (1.25 µg; n = 11) compared with saline (n = 10) in the NRM. B, time course of the increase in PWL produced by microinjection of the highest effective dose of the 1:273 combination (4.6 ng DAMGO + 1250 ng DELT; n = 5), 1:1 combination (10 ng of DAMGO + 10 ng of DELT; n = 5) or 10:1 dose combination (15 ng of DAMGO + 1.5 ng of DELT; n = 8) in the NRM. BL, baseline response latency. Arrow denotes time of microinjection. *, p < 0.05; **, p < 0.01 compared with saline at the corresponding time point (two-way repeated measures analysis of variance with Newman-Keuls test).

Figure 4 illustrates the dose-response relationships for DAMGO alone, DELT alone, and for the 1:273, 1:1, and 10:1 fixed dose ratio combinations of DAMGO and DELT. Figure 3A illustrates that the experimentally derived dose-response relationships for the 1:273 fixed dose combination of DAMGO and DELT did not differ from the theoretically derived additive dose-response relationship calculated for that fixed dose ratio. This result is consistent with an additive interaction. In contrast, the experimentally determined dose-response relationship for the 1:1 fixed dose combination of DAMGO and DELT was shifted 17-fold to the left of the theoretically derived additive dose-response relationship for that fixed dose ratio (Fig. 3B). This result is consistent with a synergistic interaction. The experimentally determined dose-response relationship for the 10:1 fixed dose combination of DAMGO and DELT was similarly shifted 10-fold to the left of the theoretically derived additive dose-response relationship for that fixed dose ratio (Fig. 3C). This result is also consistent with a synergistic interaction. Table 4 lists the ED₅₀ values and 95% CL for DAMGO and DELT when microinjected singly and in combination at the three different fixed dose ratios into the RVM.

View larger version (15K): [in this window] [in a new window]   Fig. 4. Dose-response relationships of DAMGO alone (solid squares), DELT alone (solid circles), and the experimentally determined dose-response relationship for the 1:273 mixture (A), 1:1 mixture (B), and 10:1 mixture (C) of DAMGO:DELT (open squares). For comparison, each panel also depicts the corresponding theoretical dose-response relationship for an additive interaction of each agonist in the same dose ratio (dashed line). In C, the theoretical line for the 10:1 DAMGO:DELT mixture is obscured by the dose-response relationship of DAMGO alone. Symbols are the mean ± S.E.M. of determinations in 3 to 12 rats.

	Discussion

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The present results suggest that, unlike the periphery or spinal cord, the mechanisms responsible for the enhanced antinociceptive effects of MOR and DOR agonists in the RVM following inflammation do not entail an increase in the number or affinity of MOR, or increased coupling of this receptor to G_i/o in the RVM. Although the use of homogenates can obscure changes that are restricted to specific populations of neurons, there are several arguments against this possibility. For example, increases in -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunits are readily detected in homogenates of the RVM using less quantitative Western blotting methods (Guan et al., 2003). In addition, the increase in MOR that occurs only in small- and mediumsized primary afferent neurons of CFA-treated rats (Mousa et al., 2001) can be detected in homogenates of dorsal root ganglia by radioligand binding methods (Zöllner et al., 2003). The detection of DOR, whose levels in the RVM are lower than MOR, was problematic. Appreciable specific binding was not obtained with [³H]DELT in the RVM of either saline- or CFA-treated rats, nor was significant stimulation of GTPS binding obtained in either treatment condition. However, significant stimulation was obtained in the striatum, which served as a positive control. Pradhan and Clarke (2005) similarly identified only negligible binding of [¹²⁵I]DELT, which has higher specific activity, and virtually no stimulation of [³H]GTPS binding by DELT in the RVM of naive rats. Unfortunately, methods offering greater anatomical resolution proved unworkable in the RVM. Only low levels of specific binding for [³H]DAMGO could be detected by receptor autoradiography even after 8 months of exposure of material (K. T. Sykes, D. L. Hammond, and V. Seybold, unpublished data). Although preabsorption controls confirmed the specificity of the immunohistochemical labeling of MOR or DOR in the spinal cord, such was not the case at the higher concentrations of antibody required for labeling in the RVM (K. T. Sykes and D. L. Hammond, data not shown). These findings do not exclude an increased trafficking of MOR or DOR to the plasma membrane of RVM neurons in CFA-treated rats, as has been documented for DOR in the primary afferent neuron and dorsal horn neurons (Cahill et al., 2007). In fact, increased release of enkephalins in the RVM as a result of inflammation (Hurley and Hammond, 2001) could be expected to traffic DOR to the plasma membrane of RVM neurons (Bao et al., 2003).

An alternate mechanism by which the antinociceptive effects of MOR and DOR agonists are enhanced in the RVM of CFA-treated rats may involve an interaction between exogenous agonist and increased levels of endogenous peptides. Agonists of MOR and DOR can interact in an additive or synergistic manner after concurrent intrathecal (Malmberg and Yaksh, 1992; He and Lee, 1998) or intracerebroventricular (Adams et al., 1993) administration, depending on the dose ratio tested. The interactions of MOR and DOR agonists injected at two different sites in the central nervous system of the rat have also been characterized (Yeung and Rudy, 1980; Rossi et al., 1994; Hurley et al., 1999). Only one study examined their interaction within a single supraspinal nucleus (Rossi et al., 1994). Because a fixed dose ratio approach was not used, it cannot be known whether the interaction was truly additive or synergistic. The present findings indicate that DAMGO and DELT interact synergistically to produce antinociception when they are in equal proportions or when DAMGO is the predominant component and additively when DELT is the predominant component. Four mechanisms may be considered: 1) different sites and synaptic action of MOR and DOR agonists in the RVM, 2) differences in the spinal pharmacology of MOR and DOR agonists, 3) different intracellular processing, and 4) synergism resulting from the formation of opioid receptor complexes.

In the RVM, MOR-like immunoreactivity is located on somata, dendrites, and axonal varicosities (Kalyuzhny et al., 1996). Electrophysiological studies indicate that MOR agonists inhibit synaptic transmission in the RVM by both postsynaptic and presynaptic mechanisms (Pan et al., 1990; Marinelli et al., 2002; Finnegan et al., 2004). In contrast, DOR-like immunoreactivity is predominantly located on presynaptic elements in the RVM (Kalyuzhny et al., 1996; Ma et al., 2006), although the presence of postsynaptic DOR is suggested by the ability of DELT to hyperpolarize a subpopulation of RVM neurons (Marinelli et al., 2005). Because the majority of RVM neurons hyperpolarized by DELT are also hyperpolarized by DAMGO (Marinelli et al., 2005), synergism may arise from the concerted postsynaptic actions of MOR and DOR agonists on the same neuron. Synergism may also result from a direct postsynaptic inhibition by one agonist in conjunction with a presynaptic inhibition of inputs to that neuron by the other agonist.

Opioids microinjected into the RVM produce antinociception in part by releasing neurotransmitters in the spinal cord. The antinociceptive effects of DAMGO in the RVM are blocked by intrathecal administration of CGP35348, a GABA_B receptor antagonist, or methysergide, a serotonin receptor antagonist, but not by the ₂ adrenergic receptor antagonist yohimbine (Hurley et al., 2003). In contrast, the antinociceptive effects of DELT in the RVM are blocked by intrathecal CGP35348 and yohimbine, but not by methysergide, indicating GABA and norepinephrine release in the spinal cord (Hurley et al., 2003). The multiplicative interaction of MOR and DOR agonists in the RVM may therefore reflect interactions among the different neurotransmitters released in the spinal cord subsequent to activation of RVM neurons. For example, subantinociceptive doses of norepinephrine given intrathecally enhance the antinociceptive potency of intrathecal serotonin and vice versa (DeLander and Hopkins, 1987). Serotonin also enhances the actions of GABA at GABA_A receptors in acutely dissociated dorsal horn neurons (Li et al., 2000).

The interactions between MOR and DOR agonists may involve intracellular signaling and trafficking pathways. Unlike MOR, DOR is primarily sequestered in intracellular compartments in vivo, and it is mobilized to the plasma membrane upon continuous neuronal stimulation, sustained morphine treatment, or activation by DOR agonists (for review, see Cahill et al., 2007). Following agonist binding, MOR is recycled back to the plasma membrane and resensitized for ligand activation and G protein coupling, whereas DOR is transported deeper within the endocytic pathway and targeted for lysosomal degradation and down-regulation (for review, see Waldhoer et al., 2004). Synergism may occur by changes in receptor activity or receptor trafficking induced by the presence of ligands to both receptors. For example, in neurons expressing both MOR and DOR, DAMGO may facilitate the mobilization of DOR to the plasma membrane, thereby increasing the availability of this receptor to endogenous ligand. Perhaps MOR agonists extend the lifetime of DOR by decreasing the extent of DOR lysosomal degradation and down-regulation. These mechanisms, individually or collectively, could subserve the synergistic interaction between DAMGO and DELT in the RVM.

In addition to occurring as monomers, MOR and DOR can physically interact to form heterodimers that have distinct receptor and signaling pharmacology, and intracellular trafficking (George et al., 2000; Gomes et al., 2000). In transfected heterologous cells coexpressing MOR and DOR or in cells that constitutively express opioid receptors, the addition of DOR agonists and antagonists significantly increased the number of MOR binding sites and enhanced the ability of DAMGO or morphine to stimulate [³⁵S]GTPS binding (Gomes et al., 2000, 2003, 2004). Likewise, low doses of MOR-selective ligands significantly increased DOR binding and potentiated the potency and efficacy of DOR-mediated increase in mitogen-activated protein kinase phosphorylation (Gomes et al., 2000). Heterodimers of MOR and DOR exhibit lower affinity for DOR- and MOR-selective agonists [D-Pen²,D-Pen⁵]-enkephalin and DAMGO, respectively, and higher affinity for endogenous opioid peptides such as endomorphin-1 and [Leu⁵]-enkephalin (George et al., 2000). In contrast to pertussis toxin-sensitive G protein activation observed for individually expressed MOR and DOR, MOR-DOR oligomers can stimulate G protein binding and signal transduction in a pertussis toxin-insensitive manner (George et al., 2000). The existence of MOR and DOR heterodimers in the spinal cord of mice (Gomes et al., 2004) suggests that MOR-DOR heterodimers may also exist in certain neurons of the RVM and that they may up-regulate under conditions of persistent inflammatory injury.

In summary, unlike the periphery or spinal cord, the mechanisms responsible for the potentiation of the antinociceptive effects of DAMGO in the RVM do not seem to entail changes in receptor number, affinity, or coupling. Rather, the mechanism probably entails an additive or a synergistic interaction of DAMGO with endogenous DOR-preferring agonists whose levels are increased in the RVM in CFA-treated rats. With respect to the potentiation of the antinociceptive effects of DELT, the inability to detect significant receptor binding or agonist stimulation of GTPS binding does not permit changes in receptor affinity, number, or coupling to be definitively excluded. However, had such mechanisms played a critical role, one might have expected to detect appreciable binding and stimulation of GTPS binding by DELT in the RVM of CFA-treated rats. It is unlikely that the effects of DELT are potentiated due to an interaction with endogenous MOR agonists as there seems to be no increased release of endogenous MOR-preferring agonists in the RVM in CFA-treated rats (Hurley and Hammond, 2001). Attribution of the enhancement of the antinociceptive effects of DELT to increased trafficking of DOR to the plasma membrane of RVM neurons remains an attractive hypothesis.

	Acknowledgements

 
We thank Dr. Virginia Seybold for invaluable guidance and Adam Simonsen and Julie Coyne for technical assistance.

	Footnotes

 
This work was supported by the National Institutes of Health Grant R01DA06736 (to D.L.H.) and F30DA017447 (to K.T.S.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.121954.

ABBREVIATIONS: MOR, µ-opioid receptor; DOR, -opioid receptor; CFA, Complete Freund's adjuvant; DAMGO, [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin; DELT, [D-Ala²,Glu⁴]-deltorphin (deltorphin II); RVM, rostral ventromedial medulla; GTPS, guanosine 5'-O-(3-thio)triphosphate; PWL, paw withdrawal latency; NGCp, nucleus reticularis gigantocellularis pars ; CL, confidence limits.

¹ Current affiliation: Tohoku Pharmaceutical University, Department of Physiology and Anatomy, Komatsushima Aoba-ku, Sendai, Japan.

Address correspondence to: Dr. Donna L. Hammond, Department of Anesthesia, The University of Iowa, 200 Hawkins Dr. 6 JCP, Iowa City, IA 52242. E-mail: donna-hammond{at}uiowa.edu

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