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NEUROPHARMACOLOGY
Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin
Received August 13, 2004; accepted November 11, 2004.
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Abstract |
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-glucuronide-sensitive µ-opioid receptor antagonist 3-methoxynaltrexone (6.4 pmol) selectively blocked EM-2- but not EM-1-induced antianalgesia. Pretreatment with dynorphin A(1-17) antiserum reversed only EM-2- but not EM-1-induced antianalgesia. Pretreatment with antiserum against -endorphin, [Met5]enkephalin, [Leu5]enkephalin, substance P or cholecystokinin, or with 
-opioid receptor antagonist naltrindole (2.2 nmol) or 
-opioid receptor antagonist norbinaltorphimine (6.6 nmol) did not affect EM-2-induced antianalgesia. It is concluded that EM-2 selectively releases dynorphin A(1-17) by stimulation of a novel subtype of µ-opioid receptor, tentatively designated as µ3 in the vPAG to induce antianalgesia, whereas the antianalgesia induced by EM-1 is mediated by the stimulation of another subtype of µ1- or µ2-opioid receptor.
). Both EM-1 and EM-2 potently compete with µ-opioid receptor binding with no appreciable affinity with - and -opioid receptors and selectively activate µ-opioid receptor mediated G-proteins (Goldberg et al., 1998; Narita et al., 1998; Monory et al., 2000). The increases of the [35S]GTP
S binding and antinociceptive effects induced by both EM-1 and EM-2 are selectively blocked by the pretreatment with selective µ-opioid receptor antagonist -funaltrexamine or D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2, indicating that the effects are mediated by the stimulation of µ-opioid receptors.
The µ-opioid receptors have been classified into µ1- and µ2-opioid receptors (Pasternak et al., 1980; Pasternak and Wood, 1986). Recent studies indicate that the antinociceptive effect produced by EM-1 and EM-2 is mediated by the stimulation of different subtypes of µ-opioid receptors. This view is evidenced by the findings that pretreatment with µ1-receptor antagonist naloxonazine or a novel morphine-6-glucuronide-sensitive µ-opioid receptor antagonist 3-methoxynaltrexone (Brown et al., 1997) selectively attenuates EM-2- but not EM-1-produced antinociception, suggesting that the EM-2-produced antinociception is mediated by the stimulation of µ1 and a novel µ-opioid subtype receptor (Sakurada et al., 1999, 2000, 2001). Pretreatment with D-Pro2-endomorphin-1 blocks the antinociception produced by endomorphin-1 but not endomorphin-2, and pretreatment with D-Pro2-endomorphin-2 blocks the antinociception produced by endomorphin-2 but to a much less extent endomorphin-1 (Hung et al., 2002). Pretreatment with antisense deoxynucleotide against Gi2 protein blocks the antinociception induced by EM-2 but not EM-1, whereas pretreatment with antisense deoxynucleotide against Gi1 and Gi3 proteins blocks both antinociception induced by EM-1 and EM-2, indicating that µ-opioid-coupled G-proteins such as Gi2 is regulated by EM-2 but not EM-1, whereas Gi1 and Gi3 are regulated by both EM-1 and EM-2 (Sanchez-Blazquez et al., 1999). Blockade of -opioid receptors by pretreatment with norbinaltorphimine (nor-BNI) attenuates only EM-2- but not EM-1-produced antinociception. These findings strongly support the view that EM-1 and EM-2 stimulate different subtypes of µ-opioid receptors to produce their pharmacological functions. The stimulation of this EM-2-sensitive µ-opioid receptor subsequently induces the release of dynorphin A(1-17) (Dyn) acting on -opioid receptors for producing antinociception (Tseng et al., 2000; Ohsawa et al., 2001).
We have previously demonstrated that intrathecal pretreatment of mice with a small dose of EM-2 attenuates the analgesia produced by intrathecal administration of morphine; the phenomenon has been defined as antianalgesia (Wu et al., 2003). The antianalgesia induced by EM-2 against morphine-produced analgesia has been postulated to be specifically mediated by the stimulation of a novel subtype of µ-opioid receptor, tentatively designated as µ3. This view is supported by the findings that EM-2 pretreatment induces antianalgesia, which is blocked by the pretreatment with 3-methoxynaltrexone. Pretreatment with EM-2 subsequently causes the release of Dyn to induce antianalgesia.
The ventral periaqueductal gray (vPAG) area of the mesencephalon is a primary site sensitive to opioid agonists for producing analgesia (Smith et al., 1988; Yaksh et al., 1988). Microinjection of morphine into the vPAG consistently produces analgesia, which is blocked by the pretreatment with naloxone. The analgesia produced by opioid agonists from the vPAG is mediated by the activation of the spinopetal descending pain control pathways, which are initiated from the vPAG via the rostral ventromedial medulla projecting to the spinal and trigeminal dorsal horns (Basbaum and Fields, 1984). The present study was then designed to examine the antianalgesic effect of EM-2 against the analgesia produced by morphine microinjected into vPAG. The antianalgesic effect of EM-1 was also studied for comparison.
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Materials and Methods |
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Surgical Procedures. Rats were pretreated with methylatropine bromide (5 mg/kg intraperitoneal), anesthetized with pentobarbital sodium (50 mg/kg intraperitoneal), and mounted in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA). A 23-gauge stainless steel guide cannula 12 mm in length was then implanted unilaterally 3 mm down from the surface of the skull and anchored to the skull with three stainless screws and dental cement. The coordinates for placement of the cannula for vPAG microinjection were anteroposterior 1.20 mm anterior to interaural point and 0.7 mm lateral to the midline (Paxinos and Watson, 1997). After a recovery period of at least 5 days, animals were used for the experiments.
Assessment of Analgesia. Analgesic responses were measured with the tail-flick (TF) test (D'Amour and Smith, 1941). To measure the latency of the TF response, rats were gently held by hand and their tail positioned on the apparatus (model TF6; EMDIE Instrument Co., Maidens, VA). The TF response was elicited by applying radiant heat to the dorsal surface of the tail. The intensity of the heat stimulus was set to provide a predrug TF response time of 3 to 4 s. The cutoff time was set at 8 s to minimize tissue damage.
Experimental Protocol. Groups of rats were pretreated with EM-1, EM-2, or vehicle before microinjection of morphine (9 nmol), and the TF response was measured at 5, 10, 20, 30, 40, 60, 90, and 120 min thereafter. The following three experiments were performed. 1) Determine the time course and dose-response effects of the development of EM-2-induced antianalgesia against morphine-produced antinociception. Groups of rats were pretreated with EM-2 (7.0 nmol) given into the vPAG at different times (30, 45, 90, and 180 min, respectively) before microinjection of morphine (9 nmol) given into vPAG, and the antinociception produced by morphine was determined by the TF test. A similar protocol was also used to determine the dose response of EM-1-induced antianalgesia. 2) Determine the type of receptors involved in the EM-2- and EM-1-induced antianalgesia against morphine-produced analgesia. Rats were pretreated with opioid receptor antagonists, (-)-naloxone (50 min), (+)-naloxone (50 min), 3-methoxynaltrexone (25 min) (Sakurada et al., 2000), nor-BNI (24 h) (Wu et al., 2003), or naltrindole (NTI) (10 min) (Dervisogullari et al., 1996) into the vPAG before vPAG injection of EM-2 followed by vPAG injection of morphine 45 min thereafter. The TF response was then measured 20 min after morphine injection. Other groups of rats were pretreated with (-)-naloxone (50 min), 3-methoxynaltrexone (25 min), or vehicle given into the vPAG before vPAG injection of EM-1 followed by vPAG injection of morphine 45 min thereafter. 3) Determine what endogenous opioid peptides were released by EM-2 for the induction of antianalgesia. Rats were pretreated into the vPAG with antiserum against Dyn, [Met5]enkephalin, [Leu5]enkephalin, -endorphin, cholecystokinin (CCK), normal rabbit serum (NRS), or substance P given into the vPAG 1 h prior to EM-2 vPAG injection, followed by vPAG injection of morphine 45 min thereafter. The tail-flick response was then measured 20 min after morphine injection. A similar protocol using antiserum against dynorphin A(1-17) (A/S Dyn) was also used to determine whether Dyn is involved in EM-1-induced antianalgesia against morphine-produced TF inhibition from the vPAG. Previous studies with antisera to inactivate the endogenous neuropeptides have indicated that 1 h of pretreatment time and the doses used are sufficient for their specific effects (Arts et al., 1992; Tseng and Huang, 1992; Xu and Tseng, 1997; Wu et al., 2004). To test whether Dyn alone induced antianalgesia against morphine-produced analgesia, groups of rats were pretreated with different doses of Dyn (0.5, 5, and 47 fmol) (Holmes and Fujimoto, 1992) given into the vPAG 5 min prior to vPAG injection of morphine. TF response was then measured 20 min after morphine injection.
Drugs and Drug Administration. EM-2 (Tyr-Pro-Phe-Phe-NH2) and EM-1 (Tyr-Pro-Trp-Phe-NH2) were obtained from Calbiochem (San Diego, CA). Morphine sulfate, nor-BNI, and NTI were obtained from the National Institution of Drug Abuse (Baltimore, MD). 3-Methoxynaltrexone hydrochloride and naloxone were obtained from Sigma-Aldrich (St. Louis, MO). Dyn was obtained from Bachem Biosciences (King of Prussia, PA). EM-2 and EM-1 were dissolved in sterile saline solution (0.9% NaCl solution) containing 10% hydroxypropyl--cyclodextrin, and morphine sulfate, nor-BNI, NTI, and 3-methoxynaltrexone hydrochloride were dissolved in sterile 0.9% NaCl solution. Dyn was dissolved in a 0.9% saline solution containing 0.01% Triton X-100. The antiserum against Dyn, -endorphin, [Leu5]enkephalin, [Met5]enkephalin, substance P, and CCK were produced by immunization of male New Zealand White rabbits according to the method described in previous publications. The potencies and cross-immunoreactivities of these antisera have been characterized (Tseng and Huang, 1992; Wu et al., 2004). The cross-immunoreactivity between EM-1/EM-2 and A/S Dyn was found to be less than 0.001%.
Injections into the vPAG were made by hand with a 30-gauge injection needle attached to a microsyringe via polyethylene tubing. The injection needle was inserted directly into the guide cannula. Injection volume for each microinjection was 0.5 µl, and solutions were administered over a 30-s period. The injection needles were left in place for an additional 60 s to ensure complete distribution. The stereotaxic coordinates of the vPAG injection site were aimed at anteroposterior 1.20 mm anterior to interaural point, 0.7 mm lateral to the midline, and 5.8 mm down from the surface of the skull.
Histological Identification of the Injection Site. At the end of the experiments, 0.5 µl of methylene blue solution (2%) was injected into the vPAG. The rats were then sacrificed with CO2 (100%) 10 to 20 min after injection. The brains were removed, frozen, and sectioned sagittally for microscopic identification of the injection sites. The stereotaxic atlas of rats by Paxinos and Watson (1997) was used as a guide for the identification of anatomical injection sites. Only the data obtained from rats in which the injection sites were accurately identified to be in vPAG were used for further statistical analysis.
Statistical Analysis. The analgesic responses, TF latency(s), were presented as the mean ± S.E.M. One-way analysis of variance (ANOVA) followed by Dunnett's post-test, two-way ANOVA followed by Bonferroni's post-test, or Student's t test was used to test the difference between groups. Nonlinear regression model was used to fit the dose-response curve and calculate the ED50 values, and 95% confidence intervals for EM-2 and EM-1 induced antianalgesia. The GraphPad Prism software was used to perform the statistics (version 3.0; GraphPad Software, Inc., San Diego, CA).
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Results |
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Effect of vPAG Pretreatment with Different Doses of EM-2 or EM-1 on the TF Inhibition Produced by vPAG-Administered Morphine. Groups of rats were microinjected into the vPAG with saline vehicle or different doses (1.7 to 14 nmol) of EM-2, and morphine (9 nmol) was microinjected into the vPAG 45 min later. The TF response was measured at different times thereafter. Morphine (9 nmol) microinjected into the vPAG produced TF inhibition in rats pretreated with saline vehicle for 45 min; the TF inhibition developed in 5 to 10 min, reached a maximal inhibition at 20 min (7.8 ± 0.2 s), and gradually returned to preinjection control level in 120 min. EM-2 at doses 1.7 to 7.0 nmol given into the vPAG did not cause any change in TF latency observed for 45 min. However, such a pretreatment with EM-2 dose-dependently attenuated the TF inhibition produced by morphine (9 nmol). A high dose of EM-2 (14 nmol) given into vPAG produced TF inhibition; the TF inhibition developed in 2.5 to 10 min, reached a maximal level at 20 min, and returned to preinjection level at 45 min after injection. Such a treatment with a high dose of EM-2, however, did not attenuate the TF inhibition produced by morphine (9 nmol) given into vPAG (Fig. 2, A and B).
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The effect of pretreatment with EM-1 given into the vPAG on morphine-produced TF inhibition given into vPAG was also studied. Groups of rats were pretreated with 3.5 to 28 nmol of EM-1 or vehicle given into the vPAG 45 min before vPAG-administered morphine (9 nmol). EM-1 at doses 3.5 and 7.0 nmol did not cause any change in TF latency observed for 45 min, but at a high dose (14 nmol) produced TF inhibition. The TF inhibition developed in 2.5 to 10 min, reached its peak at 20 min, and returned to the preinjection level in 45 min. Such a pretreatment with EM-1 dose-dependently attenuated vPAG morphine-produced TF inhibition. The morphine-produced TF inhibition was almost completely abolished by the pretreatment with 7.0 or 14 nmol of EM-1 (Fig. 3, A and B). Thus, EM-1 was found to be equally potent as EM-2 in inducing antianalgesia in the vPAG; the ED50 values (with 95% confidence intervals) for EM-1 and EM-2 pretreatment into the vPAG were estimated to be 3.28 (1.42 to 7.6) nmol and 3.76 (1.67 to 8.5) nmol, respectively. However, EM-2 was found to be different from that of EM-1 that pretreatment with a high dose (14 nmol) of EM-2 did not attenuate the morphine-produced TF inhibition.
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Effects of vPAG Pretreatment with (-)-Naloxone, (+)-Naloxone, 3-Methoxynaltrexone, NTI, or nor-BNI on the vPAG EM-2-Induced Antianalgesia against vPAG Morphine-Produced TF Inhibition. (-)-Naloxone given intracerebrally is a short-acting µ-opioid receptor antagonist and is expected to block, with short duration, the antianalgesic effects induced by µ-opioid agonists EM-1 or EM-2, but not long enough to block the antinociceptive effect produced by morphine. The first experiment was designed to determine the optimal pretreatment time for (-)-naloxone to block EM-1- or EM-2-induced antianalgesia but not affect the morphine-produced TF inhibition. Groups of rats were injected with (-)-naloxone (55 pmol) or saline vehicle given into the vPAG 55, 65, 75, or 95 min before vPAG injection of morphine (9 nmol), and TF response was performed 20 min after injection. Pretreatment with (-)-naloxone for 55 or 65 min, but not 95 min, significantly attenuated the morphine-produced TF inhibition (Fig. 4). Ninety-five minutes of the pretreatment time for (-)-naloxone (50 min before EM-2 injection) was then chosen for the following studies.
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The experiments were undertaken to determine what type of opioid receptors are involved in EM-2-induced antianalgesia against morphine-produced TF inhibition. Pretreatment with µ-opioid receptor antagonists (-)-naloxone (55 pmol) or 3-methoxynaltrexone (6.4 pmol) effectively blocked the EM-2-induced antianalgesia and restored the morphine-produced TF inhibition. However, pretreatment with (+)-naloxone (55 pmol), a nonopioid enantiomer of (-)-naloxone, did not affect the attenuation of the morphine-produced TF inhibition induced by EM-2 pretreatment. Pretreatment with selective -opioid receptor antagonist NTI (2.2 nmol) and -opioid receptor antagonist nor-BNI (6.6 nmol) did not affect the antianalgesia induced by EM-2 compared with the vehicle-pretreated group (Fig. 5A).
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Effects of vPAG Pretreatment with (-)-Naloxone or 3-Methoxynaltrexone on the vPAG EM-1-Induced Antianalgesia against vPAG Morphine-Produced TF Inhibition. The experiments were undertaken to determine whether the antianalgesia induced by EM-1 against morphine-produced TF inhibition is mediated by the same subtype of µ-opioid receptor as that of EM-2. Pretreatment with (-)-naloxone (55 pmol) effectively blocked the antianalgesia induced by EM-1 and restored the morphine-produced TF inhibition; however, pretreatment with 3-methoxynaltrexone (6.4 pmol) did not affect the attenuation of morphine-produced TF inhibition induced by EM-1 pretreatment (Fig. 5B).
Effects of vPAG Pretreatment with Antiserum against Dyn, [Met5]Enkephalin, [Leu5]Enkephalin, -Endorphin, CCK, Substance P, or NRS on the vPAG EM-2-Induced Antianalgesia against vPAG Morphine-Produced TF Inhibition. Groups of rats were pretreated with different doses of A/S Dyn (50, 100, 150, or 200 µg) given into the vPAG 1 h before administration of EM-2 (7.0 nmol), and morphine was administered 45 min afterward. The TF response was measured 20 min thereafter. Pretreatment with A/S Dyn dose-dependently blocked the EM-2-induced antianalgesia and enhanced the morphine-produced TF inhibition. Rats pretreated with NRS or A/S Dyn (200 µg) alone followed by vehicle injection did not affect the morphine-produced TF inhibition (Fig. 6). Pretreatment with antiserum against [Met5] enkephalin, [Leu5]enkephalin, -endorphin, CCK, or substance P given into the vPAG did not affect the attenuation of morphine-produced TF inhibition induced by EM-2 pretreatment (Table 1).
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Effects of vPAG Pretreatment with A/S Dyn on the vPAG EM-1-Induced Antianalgesia against vPAG Morphine-Produced TF Inhibition. Groups of rats were pretreated with 200 µg of A/S Dyn given into vPAG 1 h before vPAG administration of EM-1 (14 nmol), and morphine was then administered into the vPAG 45 min thereafter. The TF response was measured 20 min after morphine injection. Pretreatment with A/S Dyn did not affect the EM-1-induced antianalgesia against morphine-produced TF inhibition (Fig. 6).
Effect of vPAG Pretreatment with Different Doses of Dyn on the TF Inhibition Produced by vPAG-Administered Morphine. Since the results of the experiments described above strongly indicate that the antianalgesia induced by EM-2 is mediated by the release of Dyn, the experiment was then taken to determine whether pretreatment with Dyn peptide will mimick the effect of EM-2 and produce antianalgesia against morphine-produced TF inhibition. Groups of rats were pretreated with 0.5, 5, or 47 fmol of Dyn or saline given into the vPAG 5 min before administration of morphine (9 nmol) given into the vPAG, and the TF response was measured 20 min thereafter. Pretreatment with Dyn dose-dependently attenuated morphine-produced TF inhibition (Fig. 7).
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Discussion |
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; Zadina et al., 1997; Goldberg et al., 1998; Narita et al., 1998). However, Sakurada et al. (1999) report that pretreatment with µ1-opioid receptor blocker naloxonazine given systemically blocks the antinociception produced by intracerebroventricular or intrathecal administration of EM-2, but not EM-1, indicating that EM-1 and EM-2 may produce antinociception through the stimulation of the µ2- and µ1-opioid receptors, respectively. More recent results intimate that EM-1 and EM-2 might act on different subtypes of µ-opioid receptors other than classical µ1 and µ2 receptors. Sakurada et al. (2000) report that a novel morphine 6-glucuronide-sensitive µ-opioid receptor antagonist 3-methoxynaltrexone blocks the antinociception produced by EM-2 but not EM-1, whereas -funaltrexamine inhibits both. In the present study, pretreatment with EM-2 or EM-1 into the vPAG dose-dependently attenuated the analgesia produced by morphine given into the vPAG. The antianalgesia induced by EM-2 or EM-1 was blocked by the pretreatment with µ-opioid receptor antagonist (-)-naloxone but not by its nonopioid enantiomer (+)-naloxone, indicating that the effects are mediated by the stimulation of µ-opioid receptors. The µ-opioid receptor antagonist 3-methoxynaltrexone, which selectively blocks EM-2- and morphine-6-glucuronide-produced antinociception but not morphine antinociception (Brown et al., 1997; Sakurada et al., 2000; Pasternak, 2004) was used to determine whether it blocks the EM-2- and EM-1-induced antianalgesia. We found that 3-methoxynaltrexone pretreatment selectively blocked the EM-2- but not EM-1-induced antianalgesia. The finding is consistent with previous reports with the studies of EM-2- and EM-1-produced antinociception (Sakurada et al., 2000; Wu et al., 2003), indicating that that the antianalgesia induced by EM-2 is mediated by the stimulation of a novel subtype of µ-opioid receptor, tentatively designated as µ3, which is different from µ1- and µ2-opioid receptors stimulated by EM-1, morphine, and other µ-opioid agonists. It is likely that both antinociception and antianalgesia produced by EM-2 is mediated by stimulation of this tentative µ3-opioid receptor. Whether this newly defined µ3-opioidis belonged to any splice variant of µ-opioid receptor-1 (Pan et al., 2001) is not clear at this point.
The evidence that EM-1 and EM-2 stimulate different subtypes of µ-opioid receptors to induce their pharmacological actions can also be obtained from our previous studies of antinociceptive effects produced by EM-1 and EM-2. Intrathecal pretreatment with antisense oligodeoxynucleotides against exon-1, -4, or -8 of µ-opioid receptor clone to knockdown different isoforms of the µ-opioid receptor, differentially attenuates the antinociception produced by EM-1 and EM-2 (Wu et al., 2002). These findings strongly indicate that different subtypes of µ-opioid receptors are involved in the pharmacological actions produced by EM-1 and EM-2.
The Antianalgesia Induced by EM-2 but Not EM-1 Is Mediated by the Release of Dyn. Rady and colleagues (2001) have shown that Dyn administered intrathecally attenuates intrathecal morphine-produced analgesia. We found that vPAG pretreatment with A/S Dyn, which neutralizes dynorphin action, blocked the vPAG EM-2-induced antianalgesia. This finding is in line with a previous finding that intrathecal pretreatment with A/S Dyn blocks the intrathecal EM-2-induced antianalgesia (Wu et al., 2003). The attenuation of morphine-produced analgesia induced by EM-1 was not affected by the pretreatment with A/S Dyn, indicating that Dyn is not involved in EM-1-induced antianalgesia. These findings indicate that EM-2 but not EM-1 may release Dyn to induce antianalgesia. Furthermore, Dyn given into vPAG was found to mimic the effect of EM-2 and attenuate the morphine-produced analgesia. This is in line with previous studies that intrathecal administration of Dyn induces an antianalgesic action against intrathecally administered morphine (Holmes and Fujimoto, 1992; Aksu et al., 1993; Rady and Fujimoto, 2001). We have recently found that EM-2 but not EM-1 intrathecally perfused for 3 min dose-dependently increases the release of immunoreactive Dyn in the spinal perfusates in urethane-anesthetized rats (Leitermann et al., 2003). The increase of the release of Dyn induced by EM-2 is also blocked by (-)-naloxone and 3-methoxynaltrexone. Thus, our studies with A/S Dyn clearly indicate that Dyn is involved in EM-2- but not EM-1-induced antianalgesia. The exact neural mechanism for EM-1-induced antianalgesia is not clear. It is possible that EM-1 pretreatment may cause endocytosis and internalization of µ-opioid receptors and attenuates the morphine-produced analgesia (McConalogue et al., 1999)
Both Analgesia and Antianalgesia Produced by EM-2 Are Mediated by the Release of Dyn. Dyn released by EM-2 appears to produce biphasic effects, analgesia (Tseng et al., 2000; Ohsawa et al., 2001; Sakurada et al., 2001), and antianalgesia. This view is supported by the present finding that unlike EM-1, which induced antianalgesia even at a high dose, pretreatment with a high dose of EM-2, which itself produced antinociception, failed to attenuate the morphine-induced antinociception (Figs. 2 and 3). It is reasonable to believe that EM-2 even at high doses also induces antianalgesia, but the effect is masked by the analgesic effect of Dyn released by EM-2. The antianalgesia induced by EM-2 from vPAG is not mediated by the stimulation of - or -opioid receptors. This is evidenced by the finding that pretreatment with -opioid receptor antagonist NTI or -opioid receptor antagonist nor-BNI given into the vPAG did not reverse the attenuation of morphine-produced TF inhibition induced by EM-2 pretreatment. The finding is in line with our previous report showing that the blockade of - and -opioid receptors in the spinal cord by i.t. pretreatment with NTI and nor-BNI, respectively, do not block i.t. EM-2-induced antianalgesia (Wu et al., 2003).
We have previously reported that pretreatment with nor-BNI or A/S Dyn given spinally or supraspinally partially, but significantly, blocks the antinociception produced by EM-2 given spinally or supraspinally, indicating that the released Dyn by EM-2 subsequently stimulates -opioid receptors to produce antinociception (Tseng et al., 2000; Ohsawa et al., 2001). Thus, Dyn released by EM-2 may produce biphasic responses; an initial release of the Dyn producing analgesia, which is mediated by the stimulation of -opioid receptors, whereas a delayed release of Dyn induces antianalgesia, which is not mediated by the stimulation of -opioid receptors. Our finding is in line with previous studies that Dyn stimulates neuronal cells via a nonopioid, non-N-methyl-D-aspartate mechanism (Millan, 1999; Tang et al., 2000; Wang et al., 2001).
The Antianalgesia Induced by EM-2 Is Not Mediated by the Release of [Met5]Enkephalin, [Leu5]Enkephalin, -Endorphin, CCK, or Substance P. Intrathecal pretreatment with CCK or [Leu5]enkephalin but not [Met5]enkephalin attenuates intrathecal morphine-produced analgesia. The antianalgesic effect induced by CCK can be blocked by antiserums against CCK (1:1000 i.t., 1 h) and [Leu5]enkephalin (200 µg i.t., 1 h), respectively (Rady et al., 2001). We found in the present study that pretreatment with antiserum against [Met5]-enkephalin, [Leu5]enkephalin, or -endorphin given into the vPAG at the dose sufficient to inactivate endogenous peptides did not block the EM-2-induced antianalgesia. This result indicates that [Met5]enkephalin, [Leu5]enkephalin, and -endorphin are not involved in EM-2-induced antianalgesia.
It has been suggested that CCK and substance P may be involved in antianalgesia against morphine-produced analgesia. Tortorici et al. (2002) showed that CCK has antiopioid activity in the PAG and that tolerance to morphine in the PAG can be prevented or restored if pretreated with the nonselective CCK receptor antagonist proglumide. Substance P can be released not only from the spinal cord but also from brain regions that are involved in endogenous pain suppression (Basbaum and Fields, 1984). The findings that substance P microinjection into the PAG produces a naloxone-reversible analgesia (Mohrland and Gebhart, 1979) suggest that substance P within the brain may play a neuromodulatory role in transmitting nociceptive information. Arts et al. (1992) demonstrated that substance P given intrathecally attenuates morphine-produced analgesia indicating that substance P is also antianalgesic. We found in the present study that the vPAG pretreatment with antiserum against CCK or substance P did not affect the EM-2-induced antianalgesia, indicating that CCK and substance P are not involved in the EM-2-induced antianalgesia from vPAG.
It is concluded that pretreatment with EM-2 or EM-1 given into the vPAG dose- and time-dependently induces antianalgesia against morphine-produced analgesia. The antianalgesic effects induced by EM-2 and EM-1 from vPAG is mediated by the stimulation of different subtypes of µ-opioid receptors. The antianalgesia induced by EM-2 from vPAG is mediated by the release of Dyn acting on nonopioid receptors. [Met5]-Enkephalin, [Leu5]enkephalin, -endorphin, CCK, and substance P are not involved in EM-2-induced antianalgesia.
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Footnotes |
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ABBREVIATIONS: EM-1, endomorphin-1; EM-2, endomorphin-2; [35S]GTPS, guanosine 5'-O-(3-[35S]thio)triphosphate; nor-BNI, norbinaltorphimine; Dyn, dynorphin A(1-17); vPAG, ventral periaqueductal gray; TF, tail-flick; NTI, naltrindole; CCK, cholecystokinin; NRS, normal rabbit serum; A/S Dyn, antiserum against dynorphin A(1-17); ANOVA, analysis of variance.
Address correspondence to: Dr. Leon F. Tseng, Department of Anesthesiology, Medical College of Wisconsin, Medical Education Building, Room M4308, 8701 Watertown Plank Rd., Milwaukee, WI 53226. E-mail: ltseng{at}post.its.mcw.edu
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References |
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