Interaction Between Medullary and Spinal d1 and d2 Opioid Receptors in the Production of Antinociception in the Rat

Previous work supports the existence of two types of d opioid receptor (d1 and d2) and a role of both subtypes in the spinal cord and the ventromedial medulla (VMM) in the production of antinociception. Although it is well established that spinal and supraspinal m opioid receptors interact in a synergistic manner to produce antinociception, little is known about the interaction of d opioid receptors. This study used isobolographic analysis to determine how d1 and d2 opioid receptors in the VMM interact with their respective receptors in the spinal cord to produce antinociception. Concurrent administration of the d1 opioid receptor agonist [D-Pen,D-Pen]enkephalin at spinal and supraspinal sites in a fixed-dose ratio produced antinociception in an additive manner in the tail-flick test. In contrast, concurrent administration of very low doses of the d2 opioid receptor agonist [D-Ala,Glu]deltorphin at spinal and medullary sites produced antinociception in a synergistic manner. However, as the total dose of [D-Ala,Glu]deltorphin increased, this interaction converted to additivity. These observations suggest that different mechanisms mediate the antinociceptive effects of different doses of d2 opioid receptor agonists. The difference in the nature of the interaction produced by d1 and d2 opioid receptor agonists provides additional evidence for the existence of different subtypes of the d opioid receptor. These results also suggest that d2 opioid receptor agonists capable of crossing the blood-brain barrier will be more potent or efficacious analgesics than d1 opioid receptor agonists after systemic administration. Studies in both the mouse (Roerig and Fujimoto, 1989) and the rat (Yeung and Rudy, 1980; Siuciak and Advokat, 1989; Miyamoto et al., 1991) indicate that the concurrent administration of m opioid receptor agonists at supraspinal and spinal sites produces antinociception in a synergistic manner. This synergistic interaction is thought to be responsible for the potent antinociception produced by systemically administered m opioid receptor agonists, such as morphine. Although there is good agreement that supraspinal and spinal m opioid receptor agonists interact synergistically, there is less consensus about the manner in which supraspinal and spinal d opioid receptor agonists interact. Concurrent i.c.v. and intrathecal (i.t.) administrations of d opioid receptor agonists such as [D-Ala,D-Leu]enkephalin (Roerig et al., 1991) or [D-Pen,D-Pen]enkephalin (DPDPE) (Roerig and Fujimoto, 1989) produce antinociception in an additive manner in the radiant-heat tail-flick test in the mouse. In contrast, i.c.v. and i.t. administration of DPDPE produces antinociception in a synergistic manner in a test of mechanical nociception in the rat (Miaskowski and Levine, 1992; Miaskowski et al., 1993). Since these studies were conducted, two subtypes of the d opioid receptor, d1 and d2, have been described pharmacologically (Hammond, 1993; Porreca and Burks, 1993; Zaki et al., 1996). Both subtypes are implicated in the modulation of nociception in the spinal cord (Stewart and Hammond, 1993; Hammond et al., 1995) and the brain stem (Ossipov et al., 1995; Thorat and Hammond, 1997) of the rat. Although the earlier studies with DPDPE suggest that supraspinal and spinal d1 receptor agonists interact in an additive or a synergistic manner to produce antinociception, nothing is known about the manner in which d2 opioid receptor agonists interact. A better understanding of how d opioid receptor subtypeselective agonists interact at supraspinal and spinal sites could facilitate the development of systemically bioavailable d opioid receptor agonists as analgesics. The present study was therefore undertaken to determine how d1 and d2 opioid receptors in the ventromedial medulla (VMM) interact with their respective receptor subtype in the spinal cord to produce antinociception. An isobolographic analysis was conReceived for publication August 11, 1998. 1 This work was supported by U.S. Public Health Service Grants R01DA06736 (to D.L.H.), T32-HD07009 and F30-DA05784 (to R.W.H.), and R01DA09793 (to R.J.T.). ABBREVIATIONS: DELT, [D-Ala,Glu]deltorphin; DPDPE, [D-Pen,D-Pen]enkephalin; NRM, nucleus raphe magnus; NGCpa, nucleus reticularis gigantocellularis pars a; VMM, ventromedial medulla; i.t., intrathecal; SNC80, (1)-4-[(aR)-a-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide; TAN67, 2-methyl-4-aa(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aa-octahydro-quinolino[2,3,3-g]isoquinoline. 0022-3565/99/2892-0993$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 289, No. 2 Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 289:993–999, 1999 993 at A PE T Jornals on M ay 1, 2017 jpet.asjournals.org D ow nladed from ducted in which either the d1 opioid receptor agonist DPDPE or the d2 opioid receptor agonist DELT was administered concurrently to the VMM and the spinal cord in a fixed dose-ratio that approximated their respective ED50 values at each site. Alterations in nociceptive threshold were determined by the tail-flick and hot-plate tests. Materials and Methods These experiments were approved by the Institutional Animal Care and Use Committee of the University of Chicago. All procedures were conducted in accordance with the “Guide for Care and Use of Laboratory Animals” published by the National Institutes of Health and the ethical guidelines of the International Association for the Study of Pain. Animals. Male Sprague-Dawley rats (Sasco, Kingston, NY) weighing 300 to 350 g were anesthetized with halothane and prepared with an i.t. catheter that terminated at the L4 or L5 segment of the spinal cord (Yaksh and Rudy, 1976). Rats that exhibited motor impairments such as hindlimb or forepaw paresis were euthanized. Five to 6 days later, the rats were reanesthetized with a mixture of ketamine hydrochloride (85 mg/kg i.p.) and xylazine (9 mg/kg i.p.) and implanted with an intracerebral guide cannula (26 gauge; Plastic One, Inc., Roanoke, VA) that terminated 3 mm dorsal to either the nucleus raphe magnus (NRM) or the nucleus reticularis gigantocellularis pars a (NGCpa) in the VMM. The cannula was secured to the skull with stainless steel screws and dental acrylic. A 30-gauge stainless steel stylet was placed in the guide cannula to maintain its patency. Rats were housed individually after surgery under a 12-h light/dark cycle with food and water available ad libitum. Seven days elapsed before behavioral testing began. Rats received only one dose combination and were used only once in this study. Behavioral Tests. Nociceptive threshold was assessed by the radiant heat tail-flick and 55°C hot-plate tests. In the tail-flick test (D’Amour and Smith, 1941), the rat’s blackened tail was positioned under an intense light beam, and the time for the rat to remove its tail from the thermal stimulus was recorded. This test was performed twice at each time point on two different regions of the distal tail. The results of the two trials were averaged and recorded as the tail-flick latency. In the event that the rat did not withdraw its tail from the stimulus by 14 s, the test was terminated to prevent tissue damage, and the rat was assigned this cutoff latency. In the hot-plate test (Woolfe and MacDonald, 1944), the rat was placed on an enclosed copper plate heated to 55°C. The time between placement of the rat on the hot-plate and the occurrence of either a hindpaw lick or a jump off the surface was recorded as the hot-plate latency. Hot-plate latency was measured once per time period. In the absence of a hindpaw lick or a jump by 40 s, the test was terminated to prevent tissue damage, and this cutoff latency was assigned. Motor function was evaluated using the inclined-plane test (Rivlin and Tator, 1977). The tail-flick, inclined-plane, and hot-plate tests were performed in succession. Experimental Design. Measurements of nociceptive threshold and motor competency were made before the injection of drug. Those rats that responded in #5.0 s on the tail-flick test and #15.0 s on the hot-plate test and had inclined-plane angles of $40 degrees were used in this study. Mean baseline tail-flick and hot-plate latencies among the different dose treatment groups ranged from 3.5 to 4.0 s and from 7.9 to 12.5 s, respectively. After baseline nociceptive threshold was determined, DPDPE (0.49 ng to 4.9 mg) was microinjected into the VMM followed 25 min later by an i.t. injection of DPDPE (2.3 ng to 23 mg). The intracerebral and i.t. doses of DPDPE were administered in a fixed ratio of 1:4.7 that approximated the ratio of the ED50 values in mass units (i.e., nanograms or micrograms) of DPDPE at medullary and spinal sites, respectively. Tailflick latency, hot-plate latency, and inclined-plane angle were then redetermined 45 and 60 min after the intracerebral injection. A similar paradigm was used to characterize the interaction of DELT. After determination of baseline nociceptive threshold, DELT (0.023 ng to 0.94 mg) was microinjected into the VMM, followed 10 min later by an i.t. injection of DELT (0.063 ng to 2.51 mg). The intracerebral and i.t. doses of DELT were administered in a fixed ratio of 3:8 based on the ratio of the ED50 values of DELT at medullary and spinal sites, respectively. Tail-flick latency, hot-plate latency, and inclinedplane angle were then redetermined 30 and 40 min after the intracerebral injection. This order of drug administration ensured that the peak effects of DPDPE (Hammond et al., 1995; Thorat and Hammond, 1997) or DELT (Stewart and Hammond, 1993; Thorat and Hammond, 1997) at medullary and spinal sites would coincide and encompass both testing times. The agonists were administered in a fixed-dose ratio to allow characterization of the interaction between medullary and spinal sites by the isobolographic method (Roerig and Fujimoto, 1988, 1989; Tallarida et al., 1989; Tallarida, 1992b). Data on the hot-plate test were, by default, obtained at the dose-ratio determined for the tail-flick test because neither DPDPE nor DELT increased hot-plate latency in a dose-dependent manner after microinjection in the medulla. It was therefore not possible to calculate a ratio of ED50 values to be administered in the hot-plate test. Statistical Analysis. A two-way ANOVA for repeated measures was used to compare the effects of DPDPE or DELT with those of the vehicle control. The Newman-Keuls test was used for post-hoc comparisons among the individual group mean values. Dose-response relationships for DPDPE or DELT at each site alone or in combination were determined using the individual tail-flick and hot-plate latencies obtained at the time of peak effect. For concurrent injection, the dose was expressed as the total dose (VMM 1 i.t.) of drug administered. The ED50 value was defined as the dose that produced the half-maximal possible increase in response latency. This value corresponded to 9.0 s in the tail-flick test and 25.0 s in the hot-plate test. Fieller’s theorem as applied by Finney (1964) was used to determine the 95% confidence limits. The experimentally derived dose-response relationship for the total dose of drug was then compared with its theoretical dose-additive relationship by standard parallel line assay methods (Finney, 1964; Tallarida et al., 1989; Tallarida, 1992b). In addition, an isobologram was constructed using the ED50 values for each agonist for the individual VMM and i.t. administrations and their 95% confidence limits (Tallarida et al., 1989). The experimentally derived ED50 value of the agonist combination was then plotted on the isobologram and statistically compared with the theoretical dose-additive point (Tallarida, 1992b). Data on the effects of DPDPE (Hammond et al., 1995) and DELT (Stewart and Hammond, 1993) at i.t. or medullary (Thorat and Hammond, 1997) sites of injection were taken from previous studies published by personnel from this laboratory. Because the data on the antinociceptive potency of DPDPE or DELT in the VMM were obtained at about the same time as this study was conducted, replication of these dose-effect curves was not necessary. The data on the effects of i.t. administered DPDPE and DELT were obtained several years earlier. However, two independent estimates of the ED50 values of both DPDPE and DELT conducted several years apart yielded ED50 values for each drug that were not significantly different (Stewart and Hammond, 1993; Hammond et al., 1995). Given that the same sources of drug and animals were used in all studies, a third replication of the dose-effect curves of i.t. administered DPDPE or DELT was not justified. Histology. At the conclusion of testing, the rats were euthanized by CO2 inhalation. The location and patency of the i.t. catheter were determined by direct visual inspection after a laminectomy and an i.t. injection of India ink. The brains were removed and fixed by immersion in a 4% formaldehyde and 30% sucrose solution. Transverse sections of the brain stem (25 mm) were cut on a cryostat microtome and stained with cresyl violet. The location of each microinjection site was plotted on transverse sections of the rat brain 994 Medullary and Spinal Interaction of d Opioid Agonists Vol. 289 at A PE T Jornals on M ay 1, 2017 jpet.asjournals.org D ow nladed from stem modified from those provided by Neurographics (Kanata, Ontario) and was verified by a person unaware of the treatment. Drugs. DPDPE (lot no. 116H58302) and DELT (lot no. 44H08641) were purchased from Sigma Chemical Co. (St. Louis, MO) and dissolved in saline. Intracerebral microinjections were made over a 60to 120-s period in a volume of 0.4 ml via a 33-gauge stainless steel injector that extended 3 mm beyond the tip of the guide cannula. After injection, the cannula was left in place for an additional 60 s to allow the drug to diffuse locally and to limit its diffusion up the injection track. Intrathecal injections were made over a 60-s period in a volume of 10 ml and were followed by 10 ml of saline to flush the catheter. The progress of drug delivery to supraspinal and spinal sites was monitored by the movement of an air bubble in the polyethylene tubing that connected the injector to the syringe pump.