JPET Fast Forward. Published on October 13, 2003 as DOI: 10.1124/jpet.103.056242 JPET FastThis articleForward. has not Published been copyedited on and October formatted. 13,The final2003 version as DOI:10.1124/jpet.103.056242 may differ from this version.
Dynorphinergic mechanism mediating endomorphin-2-induced
anti-analgesia in the mouse spinal cord
Hsiang-En Wu, Han-Sen Sun, Moses Darpolar, Randy J. Leitermann,
John P. Kampine and Leon F. Tseng*
Department of Anesthesiology, Medical College of Wisconsin, Downloaded from
Milwaukee, WI 53226, USA
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Copyright 2003 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on October 13, 2003 as DOI: 10.1124/jpet.103.056242 This article has not been copyedited and formatted. The final version may differ from this version.
a) Running title: Endomorphin-2-induced anti-analgesia b) Corresponding author:
Leon F. Tseng, Ph.D.
Department of Anesthesiology
Medical College of Wisconsin
Medical Education Building, Room M4308
8701 Watertown Plank Road Downloaded from
Milwaukee, WI 53226
Tel: (414) 456-5686, jpet.aspetjournals.org Fax: (414) 456-6507
E-mail: [email protected] c) The number of text pages: 31 at ASPET Journals on September 26, 2021
The number of figures: 7
The number of table: 1
The number of references: 38
The number of words in Abstract: 259
The number of words in Introduction: 462
The number of words in Discussion: 1314 d) Abbreviations:
Dyn, Dynorphin A(1-17); EM-1, endomorphin-1; EM-2, endomorphin-2; CCK,
cholecystokinin; DAMGO, [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin; NTI, naltrindole;
nor-BNI, nor-binaltorphimine; NRS, normal rabbit serum; TF, Tail-flick response;
%MPE, percent maximum possible effect
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e) Recommended section
Neuropharmacology
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Abstract:
We have previously demonstrated that both endomorphin-1 (EM-1) and endomorphin-2 (EM-2) at high doses (1.75- 35 nmol) given intrathecally (i.t.) or intracerebroventricularly produce antinociception by stimulation of µ-opioid receptors.
Now, we report that EM-2 at small doses (0.05-1.75 nmol), which injected alone did not produce antinociception, produces anti-analgesia against opioid agonists-induced antinociception. The tail-flick (TF) response was used to test the antinociception in male Downloaded from
CD-1 mice. Intrathecal pretreatment with EM-2 (0.02-1.75 nmol) 45 min prior to i.t. morphine (3.0 nmol) injection dose-dependently attenuated morphine-induced TF jpet.aspetjournals.org inhibition. On the other hand, a similar dose of EM-1 (1.64 nmol) failed to produce any anti-analgesic effect. The EM-2 (1.75 nmol)-produced anti-analgesia against morphine-
µ induced TF inhibition was blocked by i.t. pretreatment with the -opioid antagonist at ASPET Journals on September 26, 2021 naloxone or 3-methoxynaltrexone, but not δ-opioid receptor antagonist naltrindole, κ- opioid receptor antagonist nor-binaltorphimine, or NMDA receptor antagonist MK-801.
The EM-2-induced anti-analgesic effect against morphine-induced TF inhibition was blocked by i.t. pretreatment with antiserum against dynorphin A (1-17), but not β- endorphin, [Met]-enkephalin, [Leu]-enkephalin, or cholecystokinin antiserum (200 µg each). The i.t. EM-2 pretreatment also attenuated the TF inhibition induced by other µ- opioid agonists, [D-Ala2, N-Me-Phe4, Gly-ol5]enkephalin, EM-1 and EM-2, δ-opioid agonist deltorphin II and κ-opioid agonist U50,488H. It is concluded that EM-2 at sub- analgesic doses presumably stimulates a subtype of µ-opioid receptor and subsequently induces the release of dynorphin A(1-17) to produce anti-analgesic effects against µ-, δ- or κ-agonists-induced antinociception. The EM-2-induced anti-analgesia is not mediated
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by the release of [Met]-enkephalin, [Leu]-enkephalin, β-endorphin or cholecystokinin, nor does it involve κ- or δ-opioid or NMDA receptors in the spinal cord.
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Endogenous opioid tetrapeptides, endomorphin-1 (EM-1) and endomorphin-2
(EM-2), have been found to be highly selective for µ-opioid receptors (Zadina et al.,
1997). Both EM-1 and EM-2 potently compete with µ-opioid receptor binding with no appreciable affinity with δ- and κ-opioid receptors and selectively activate µ-opioid receptor mediated G-proteins with [35S]GTPγS binding (Goldberg et al., 1998; Monory et al., 2000; Narita et al., 1998, 2000). The increases of the [35S]GTPγS binding and Downloaded from antinociceptive effects induced by both EM-1 and EM-2 are selectively blocked by the pretreatment with selective µ-opioid receptor antagonist, β-funaltrexamine or D-Phe-Cys-
Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2, indicating that the effects are mediated by the jpet.aspetjournals.org stimulation of µ-opioid receptors.
However, recent studies indicate that the antinociceptive effects induced by EM-1 at ASPET Journals on September 26, 2021 and EM-2 are mediated by the stimulation of different subtypes of µ-opioid receptors.
Pretreatment with µ-opioid receptor antagonist, 3-methoxynaltrexone, selectively attenuated EM-2- but not EM-1-induced antinociception, where β-funaltrexamine inhibits both (Sakurada et al., 2000). There is an asymmetric cross-tolerance between EM-1 and
EM-2, where mice made acute tolerant to EM-1 are not cross-tolerant to EM-2, but mice made acute tolerant to EM-2 cause partial cross-tolerance to EM-1 (Wu et al., 2001).
Intrathecal (i.t.) pretreatment with antisense oligodeoxynucleotides against exon-8 of µ- opioid receptor clone inhibits the antinociception induced by EM-1 but not EM-2 (Wu et al., 2002b). These findings strongly support the view that EM-1 and EM-2 stimulate different subtypes of µ-opioid receptors to produce their pharmacological functions.
Dyn has been shown to process both antinociceptive and pro-nociceptive actions
(Millan, 1999 for review). We found in our previous studies that the TF inhibition
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induced by EM-2, but not EM-1 or other µ-opioid agonists given i.c.v. or i.t., is attenuated by the pretreatment with antiserum against dynorphin A(1-17) (Dyn) or κ- opioid antagonist nor-binaltorphimine (nor-BNI), indicating that the EM-2-induced antinociception is mediated in part by the release of Dyn acting on κ-opioid receptors
(Tseng et al., 2000; Ohsawa et al., 2001). Early publications have provided evidence that one of the anti-analgesic actions against morphine- and other opioids-induced analgesia is mediated by the release of Dyn (Fujimoto et al., 1990; Holmes and Fujimoto, 1992, 1993; Downloaded from
Art et al., 1992; Aksu et al., 1993). In the present study, we have demonstrated another aspect of the action of EM-2. Pretreatment with a sub-analgesic dose of EM-2 attenuates jpet.aspetjournals.org the antinociception induced by subsequent injection of morphine or other opioids. We propose that the anti-analgesia of EM-2 is mediated by the stimulation of a subtype of µ-
opioid receptors and the release of Dyn. Experiments were performed to demonstrate at ASPET Journals on September 26, 2021 that this anti-analgesia induced by EM-2 is selectively blocked by the pretreatment with the selective µ-opioid receptor antagonist 3-methoxynaltrexone and antiserum against
Dyn. We also determined if this EM-2-induced anti-analgesia is selective against antinociception induced by µ-opioid agonists or is generalized to δ- or κ-opioid agonists.
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Materials and Methods
Animals. Male CD-1 mice weighing 25-30 g (Charles River Breeding
Laboratory, Wilmington, MA) were used. Animals were housed five per cage in a room maintained at 22 ± 0.5°C with an alternating 12-h light-dark cycle. Food and water were available ad libitum. The animals were used only once. All experiments were approved by and conformed to the guidelines of the Animal Care Committee of the Medical
College of Wisconsin. Downloaded from
Assessment of Antinociception. Antinociceptive responses were measured with the tail-flick (TF) test (D’Amour and Smith, 1941). To measure the latency of the TF response, jpet.aspetjournals.org mice were gently held with the tail put 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 pre-drug TF response time of 3 at ASPET Journals on September 26, 2021 to 4 s. The inhibition of the TF response was expressed as “percent maximum possible effect
(%MPE)”, which was calculated as [(T1-T0) / (T2-T0)] × 100. T0 and T1 were the TF latencies before and after i.t. injection of morphine, respectively, and T2 was the cutoff time, which was set at 10 s.
Experimental Protocols. Intrathecal injection was performed according to the procedure of Hylden and Wilcox (1980), using a 25-µl Hamilton syringe with a 30-gauge needle. The injection volume was 5 µl. The following experiments were performed. 1).
Groups of mice were administered i.t. with EM-1 1.64 nmol or EM-2 1.75 nmol and TF responses were measured at different time after injection for 2 h. Other groups of mice were pretreated i.t. with EM-1 1.64 nmol, EM-2 1.75 nmol, or vehicle 45 min prior to i.t. morphine (3.0 nmol) injection and the TF responses were then measured every 5-10 min
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after injection for 1 h. 2). Determine the time course and the dose-response relationship of EM-2 pretreatment for the development of anti-analgesia against morphine-induced antinociception. Groups of mice were pretreated i.t. with EM-2 (1.64 nmol) for different times (15-120 min) or different doses (0.02-17.5 nmol) of EM-2 for 45 min before i.t. administration of morphine and the TF responses were measured at different time thereafter. 3). Determine the type of receptors involved in EM-2-induced anti-analgesia
against morphine-induced TF inhibition. Mice were pretreated i.t. with the selective Downloaded from opioid antagonist, 3-methoxynaltrexone (6.4 pmol) 25 min (Sakurada et al., 2000), δ- opioid receptor antagonist naltrindole (NTI, 11.1 nmol) 10 min (Mizoguchi et al., 1995; jpet.aspetjournals.org Wu et al., 2002a), κ-opioid receptor antagonist nor-binaltorphimine (nor-BNI, 6.6 nmol)
24 h (Tseng et al., 1997; Ohsawa et al., 2001), non-selective opioid-receptor antagonist
naloxone (5.5 to 55 pmol) 10 min, or NMDA receptor antagonist MK-801 (10 nmol) 20 at ASPET Journals on September 26, 2021 min (Gardell et al., 2002) before i.t. injection of EM-2 (1.75 nmol). Morphine (3.0 nmol) was then injected i.t. 45 min after EM-2 injection and TF responses were measured at different times thereafter. 4). Determine the type of endogenous neuropeptides involved in EM-2-induced anti-analgesia against morphine-induced TF inhibition. Groups of mice were pretreated i.t. with antiserum against Dyn, cholecystokinin-8s (CCK-8s), β- endorphin, [Leu5]enkephalin, [Met5]enkephalin or normal rabbit serum (NRS) 1h prior to i.t. injection of EM-2 and morphine was then injected i.t. 45 min thereafter. The TF responses were then measured at different times after morphine injection. The doses of antiserum against Dyn were 50 to 300 µg and 200 µg for other antisera. The doses of antisera used have been shown previously to be sufficient for their specific effects
(Ohsawa et al., 2001; Tseng and Huang, 1992; Wu et al., 2002a; Xu and Tseng, 1997).
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Finally, 5) determine if the EM-2-induced anti-analgesia is selective against opioid µ agonists or is generalized to δ and κ agonists. Groups of mice were injected i.t. with EM-
2 (1.75 nmol) 45 min prior to i.t. administration of DAMGO (0.02 nmol), EM-1 (16.4 nmol), EM-2 (35 nmol), deltorphin II (6.4 nmol) or U50,488H (123.2 nmol) and the TF response were measured at different times after injection.
Drugs and Antisera. Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin- Downloaded from 2 (Tyr-Pro-Phe-Phe-NH2) were purchased from Calbiochem Corp. (La Jolla, CA). NTI and nor-BNI were obtained from National Institute of Drug Abuse (Baltimore, MD).
U50,488H, naloxone, 3-methoxynaltrexone, and MK-801 were purchased from Sigma jpet.aspetjournals.org
(St. Louis, MO). [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO) and deltorphin II were purchased from Phoenix Pharmaceuticals, Inc. (Belmont, CA). The EM-1 and EM-
2 for i.t. injection were dissolved in 0.9% saline containing 10% hydroxypropyl-beta- at ASPET Journals on September 26, 2021 cyclodextrin. U50,488H, 3-methoxynaltrexone, NTI, nor-BNI, and MK-801 were dissolved in 0.9% saline. The DAMGO was dissolved in 0.9% saline containing 0.01% of Triton X-100 and deltorphin II was dissolved in 0.9% saline containing 1% of DMSO.
The antiserum against dynorphin A (1-17), β-endorphin, [Leu5]-enkephalin, [Met5]- enkephalin, and CCK-8s were produced by immunization of male New Zealand white rabbits according to the method described in previous publications and the potencies and cross-immunoreactivity of these antisera have been characterized (Tseng and Collin,
1993; Wu et al., 2002a; Tseng and Huang, 1992).
Statistical analysis. The antinociceptive responses, percent maximum possible effect (%MPE), 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
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post-tests or Student’s t-test was used to test the differences between groups. Non-linear regression model was used to fit the dose-response curve and calculates the ED50 value and 95% confidence interval (CI) of EM-2 induced anti-analgesia. The GraphPad Prism software was used to perform the statistics (version 3.0; GraphPad Software, Inc., San
Diego, CA).
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Results:
Tail-flick responses following i.t. injection of 1.64 nmol of EM-1 or 1.75 nmol of EM-2 Groups of mice were injected i.t. with EM-1 (1.64 nmol), EM-2 (1.75 nmol), or vehicle and the TF response were measured at different times after injection. EM-1 and
EM-2 at such a dose produced a short and weak TF inhibition (23 and 15 % MPE, respectively) at 5 min and returned to vehicle control levels in 10-15 min for the rest 2 h
of measurement times (Fig. 1). Downloaded from
Effect of i.t. pretreatment with 1.64 nmol of EM-1 or 1.75 nmol of EM-2 on the TF inhibition induced by i.t.-administered morphine (3.0 nmol) Groups of mice jpet.aspetjournals.org were pretreated i.t. with 1.64 nmol of EM-1, 1.75 nmol of EM-2 or vehicle 45 min prior to i.t. injection of morphine (3.0 nmol) and the TF responses were measured at various times after injection. The morphine-induced TF inhibition was attenuated by i.t. at ASPET Journals on September 26, 2021 pretreatment with EM-2, but not EM-1 or vehicle (Fig. 2).
Effects of different times of pretreatment with EM-2 given i.t. on the TF inhibition induced by i.t.-administered morphine. Groups of mice were pretreated i.t. with EM-2 (1.75 nmol) at various times before i.t. injection of morphine (3.0 nmol), and the TF inhibition was measured 15 min after the injection. Other groups of mice pretreated i.t. with vehicle served as controls. The i.t. administration of morphine (3.0 nmol) produced 77 to 90 %MPE of the maximum TF inhibition in mice pretreated i.t. for different times with vehicle. Intrathecal pretreatment with EM-2 time-dependently attenuated the TF inhibition induced by i.t. morphine; the attenuation of the morphine- induced TF inhibition developed slowly, reached the lowest level (18.6±5.0 % MPE) at
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45 min and returned to control level at 90 or 120 min (Fig. 3A). Pretreatment time of 45 min for EM-2 was then used for the following experiments.
Effects of i.t. pretreatment with different doses of EM-2 on the TF inhibition induced by i.t.-administered morphine Groups of mice were pretreated i.t. with different doses of EM-2 (0.02-17.5 nmol) 45 min before i.t. injection with morphine (3.0 nmol) and the TF response was measured 15 min after injection. EM-2 at 0.05 to 1.75
nmol dose-dependently attenuated the TF inhibition induced by i.t. morphine (Fig. 3B). Downloaded from
The ED50 for EM-2 to produce anti-analgesia was estimated to be 0.06 nmol (95% CI:
0.02-0.15 nmol). Pretreatment dose of EM-2 at 1.75 nmol was found to produce a jpet.aspetjournals.org maximum inhibition and was therefore used for the following experiments.
Effect of i.t. pretreatment with naloxone, 3-methoxynaltrexone, NTI, nor-
BNI and MK-801 on the EM-2-produced anti-analgesia against morphine-induced at ASPET Journals on September 26, 2021
TF inhibition The experiments were designed to determine if the anti-analgesic effect of
EM-2 against i.t. morphine-induced TF inhibition is mediated by the stimulation of a subtype of µ-opioid receptor and to determine if NMDA receptors, δ- and κ-opioid receptors are involved in EM-2-induced anti-analgesia. Intrathecal pretreatment with a non-selective µ-opioid receptor antagonist naloxone (5.5-55 pmol) given 10 min before
EM-2 administration blocked the anti-analgesia produced by i.t. EM-2 pretreatment and dose-dependently restored the sensitivity to morphine (3 nmol) for producing TF inhibition (Fig. 4). Similarly, i.t. pretreatment with a selective µ-opioid receptor antagonist 3-methoxynaltrexone (6.4 pmol) given 25 min before EM-2 administration significantly blocked the anti-analgesic effect of EM-2 and enhanced the morphine- induced (3.0 nmol) TF inhibition (87 % MPE). On the other hand, i.t. pretreatment with
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the selective δ-opioid receptor antagonist NTI (11.1 nmol), κ-opioid antagonist nor-BNI
(6.6 nmol), or NMDA receptor antagonist MK-801 (10 nmol) did not affect the attenuation of the morphine-induced TF inhibition produced by EM-2 compared with vehicle pretreated groups (Fig. 5).
Effects of i.t. pretreatment with antisera against Dyn, β-endorphin, [Met]- enkephalin, [Leu]-enkephalin, or CCK-8s on the attenuation of morphine-induced
TF inhibition produced by EM-2 pretreatment Antiserum against Dyn and other Downloaded from neuropeptides were used to determine if Dyn or other neuropeptides is involved in mediating the EM-2 produced anti-analgesia against i.t.-morphine-induced jpet.aspetjournals.org antinociception. Intrathecal pretreatment with different doses (50 to 300 µg) of antiserum against Dyn dose-dependently restored the sensitivity to morphine and enhanced the
morphine-induced TF inhibition in mice pretreated i.t. with EM-2 (Fig. 6, Table 1). at ASPET Journals on September 26, 2021
However, i.t. pretreatment with antiserum (200 µg each) against β-endorphin,
[Met]enkephalin, [Leu]enkephalin, or CCK-8s did not alter the morphine-induced TF inhibition in mice pretreated with EM-2. Intrathecal pretreatment with normal rabbit serum (NRS) also showed no effect on EM-2-induced anti-analgesia against morphine- induced TF inhibition (Table 1).
Effects of i.t. pretreatment with EM-2 on the TF inhibition induced by µ-, δ- and κ-opioid agonists. Groups of mice were pretreated i.t. with EM-2 (1.75 nmol) 45 min before i.t. injection of µ-opioid receptor agonists DAMGO (0.02 nmol), EM-1 (16.4 nmol), or EM-2 (35 nmol), δ-opioid receptor agonist deltorphin II (6.4 nmol), or κ-opioid receptor agonist U50,488H (123.2 nmol) and the TF response was measured 15 min after injection. Mice pretreated i.t. with vehicle served as control. The inhibition of the TF
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response induced by DAMGO, EM-1, EM-2, deltorphin II, or U50,488H was all attenuated by i.t. pretreatment with EM-2 (1.75 nmol) (Fig. 7).
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Discussions:
EM-2 at sub-antinociceptive doses produces anti-analgesia against morphine- induced antinociception We have previously demonstrated that EM-2 at high doses (35 nmol) given i.t. produces antinociception, which is mediated by the stimulation of µ- opioid receptors (Ohsawa et al., 2001). This is evidenced by the findings that the TF inhibition induced by EM-2 given i.t. is blocked by the pretreatment with selective µ- opioid receptor antagonist β-funaltrexamine, or D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr- Downloaded from
NH2 (Goldberg et al., 1998; Ohsawa et al., 2001). We demonstrated in the present studies that EM-2 at small doses (0.05-1.75 nmol), which given i.t. alone did not produce jpet.aspetjournals.org any appreciable antinociception, but attenuated the antinociception induced by i.t.- administered morphine. This anti-analgesia of EM-2 against morphine-induced
antinociception developed slowly, reached a maximal peak at 45 min and returned to at ASPET Journals on September 26, 2021 control level 90-120 min after EM-2 pretreatment.
The anti-analgesia produced by EM-2 pretreatment is mediated by the stimulation of a subtype of µ-opioid receptor Both EM-1 and EM-2 are highly selective ligands for µ-receptors with high affinity. However, we found in the present study that only pretreatment with EM-2, but not EM-1 at the comparable pharmacological potency dose produced anti-analgesia against morphine-induced antinociception. The finding indicates that the EM-2-induced anti-analgesia is mediated by the stimulation of a subtype of µ-opioid receptor, which is only sensitive to the EM-2, but not EM-1. This view is further supported by the finding that pretreatment with 3- methoxynaltrexone blocked the anti-analgesic effect of EM-2 but not EM-1. 3-
Methoxynaltrexone is a selective µ-opioid receptor antagonist, which blocks selectively
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the antinociception induced by EM-2, but not EM-1 or morphine (Sakurada et al., 2000).
The presence of different subtypes of µ-opioid receptors for EM-1 and EM-2 to perform their pharmacological actions can also be obtained from our previous studies of antinociceptive effects induced by high doses of EM-1 and EM-2. There is an asymmetric cross-tolerance between EM-1 and EM-2 for producing antinociception.
Mice or rats made acutely tolerant to EM-1 are not cross-tolerant to EM-2, while mice or rats made tolerant to EM-2 are partially cross-tolerant to EM-1 (Wu et al., 2001, Hung et Downloaded from al., 2002). Intrathecal pretreatment with antisense oligodeoxynucleotides against exon-1 or -4 of µ-opioid receptor clone is about equally effective in attenuating the jpet.aspetjournals.org antinociception induced by EM-1 and EM-2. On the other hand, pretreatment with antisense oligodeoxynucleotides against exon-8 of µ-opioid receptor clone attenuates the
antinociception induced by EM-1, but not EM-2 (Wu et al., 2002b). Thus distinct at ASPET Journals on September 26, 2021 subtypes of µ-opioid receptors, which reflect different splice variants of exons of MOR-
1, are involved in pharmacological responses induced by EM-1 and EM-2.
The µ-opioid receptors have been pharmacologically further classified into µ1 and
µ2-opioid receptors by Pasternak and his colleagues ((Pick et al., 1991; Pasternak, 1993).
The proposed subtype of µ-opioid receptors for EM-2 to produce anti-analgesia is speculated to belong to pharmacologically identified µ1- rather than µ2-opioid receptors.
This view is supported by the findings obtained from the antinociceptive study of EM-1 and EM-2 in mice. Pretreatment of mice with µ1-opioid receptor antagonist naloxonazine is more effective in blocking the antinociception induced by i.t.-administered EM-2 than
EM-1, indicating that the antinociceptive effect induced by EM-2 is mediated by the stimulation of µ1- rather than µ2-opioid receptors. Interestingly, pretreatment with
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antiserum against Dyn or κ-opioid antagonist nor-BNI blocks the antinociception induced by i.t.-administered EM-2, but not EM-1. Thus, EM-2 at high doses stimulates µ1-opioid receptors to induce the release of Dyn acting on κ-opioid receptors for the production of antinociception (Sakurada et al., 2001; Ohsawa et al., 2001).
δ- or κ-opioid receptors or NMDA receptors are not involved in EM-2- produced anti-analgesia Pretreatment with δ-opioid receptor antagonist NTI or κ-opioid Downloaded from receptor antagonist nor-BNI did not affect morphine-induced antinociception in EM-2 pretreated mice, indicating that δ- and κ-opioid receptors may not be involved in EM-2-
induced anti-analgesia. Spinal administration of dynorphin produces a long-lasting jpet.aspetjournals.org allodynia, which cannot be blocked by pretreatment with non-selective opioid antagonist naloxone but blocked by pretreatment with NMDA receptor antagonist, MK-801
(Vanderah et al., 1996; Laughlin et al., 1997). However, Tang and colleague (2000) at ASPET Journals on September 26, 2021 demonstrate that dynorphin A elicits an increase in intracellular calcium level through a non-opioid and non-NMDA mechanism, which subsequently modulate NMDA receptor activity. Our results showed that neither nor-BNI nor MK-801 blocked EM-2-induced anti-analgesia against morphine-induced antinociception, indicating that Dyn released after EM-2 administration may act via non-opioid and non-NMDA receptor systems to produce its anti-analgesic effect.
The anti-analgesia induced by EM-2 is mediated by the release of Dyn, but not β-endorphin, [Met]-enkephalin, [Leu]-enkephalin or cholecystokinin We found in the present study that i.t. pretreatment with antiserum against Dyn, which neutralizes
Dyn action, blocked the EM-2-produced anti-analgesia and re-established the antinociception induced by i.t.-administered morphine. The finding indicates that EM-2
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may release Dyn to produce the anti-analgesic effect. We have recently found that EM-2 perfused i.t. for 3 min at (5-50 nmol) dose-dependently increases the release of immunoreactive Dyn in the spinal perfusates measured with ELISA in urethane- anesthetized rats. The increase of the release of Dyn induced by EM-2 is also blocked by naloxone (Leitermann et al., 2003). Thus, our studies with antiserum against Dyn clearly indicate that Dyn may be involved in EM-2-induced anti-analgesia against morphine-
induced antinociception. Downloaded from
Earlier, Holmes and Fujimoto (1993) find that i.t. injection with a small dose of naloxone (5 pg) or β-funaltrexamine (0.25 ng) enhances the i.t.-administered morphine- jpet.aspetjournals.org induced antinociception. Also, i.t. pretreatment with antiserum against Dyn, which neutralizes Dyn action, also enhances morphine-induced antinociception. The findings
µ indicate that there is a tonic release of a -opioid ligand, which induces the release of at ASPET Journals on September 26, 2021
Dyn to antagonize i.t. morphine-induced antinociception. Dyn function as an anti- analgesic modulator to oppose opioid-induced antinociception appears to be specific to
Dyn. Other dynorphins such as dynorphin A [1-8] , dynorphin A [2-17], dynorphin B and α and β-neoendorphin lack such anti-analgesic activity (Rady et al., 1991). We speculate that this endogenous µ-opioid ligand to induce Dyn release for producing anti- analgesia is EM-2, which is densely located in the dorsal horn of the spinal cord.
We found in the present study that pretreatment with antiserum against β- endorphin, [Met]-enkephalin or [Leu]-enkephalin did not affect the EM-2-induced anti- analgesia against morphine-induced antinociception. The results indicate that β- endorphin, [Met]-enkephalin and [Leu]-enkephalin may not be involved in EM-2-induced anti-analgesia.
19 JPET Fast Forward. Published on October 13, 2003 as DOI: 10.1124/jpet.103.056242 This article has not been copyedited and formatted. The final version may differ from this version.
Cholecystokinin has been shown to be a pro-nociceptive and anti-opioid ligand, which attenuates the antinociceptive properties of µ-opioid receptor agonists (Millan,
2002). Blockade of the action of CCK, especial CCKB receptor, potentiates morphine- induced antinociception (Singh et al., 1996; McNally, 1999). However, our result clearly demonstrated that CCK was not involved in the EM-2-induced anti-analgesia, which was evidenced by our finding that the pretreatment of antiserum against CCK could not restore morphine-induced antinociception. Downloaded from
The EM-2 pretreatment non-selectively attenuates the antinociception induced by opioid µ, δ or κ agonists. The question remains whether the EM-2-induced jpet.aspetjournals.org anti-analgesia is selective against antinociception induced by µ-opioid agonists or can be generalized to δ- and κ-opioid agonists. We found that pretreatment with EM-2
attenuated the TF inhibition induced by δ-opioid receptor agonist deltorphin II and κ- at ASPET Journals on September 26, 2021 opioid receptor agonist U50,488H, indicating that the EM-2-induced anti-analgesia can also be generalized to the antinociception induced by δ- and κ-opioid receptor agonists.
Since Dyn has been proposed to be an endogenous ligand for κ-opioid receptors, the blockade of κ-opioid opioid by nor-BNI should have blocked the EM-2-induced anti- analgesia. Since we found that the blockade of the κ-opioid receptors by nor-BNI pretreatment did not affect the EM-2-induced anti-analgesia (Figure 5), the Dyn released by EM-2 does not act on κ-opioid for producing anti-analgesia. This view is consistent with the study by Wang et al (2001) of dynorphin-mediated chronic neuropathic pain.
They reported that pronociceptive action of dynorphins for the maintenance of neuropathic pain is mediated by the activation of non-opioid receptors.
20 JPET Fast Forward. Published on October 13, 2003 as DOI: 10.1124/jpet.103.056242 This article has not been copyedited and formatted. The final version may differ from this version.
It is concluded that i.t. pretreatment with low dose of EM-2 stimulates a subtype of µ-opioid receptor to elicit the release of Dyn. The release of Dyn by EM-2 pretreatment subsequently elicits an anti-analgesic effect against µ-, δ or κ-opioid agonist-induced antinociception. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021
21 JPET Fast Forward. Published on October 13, 2003 as DOI: 10.1124/jpet.103.056242 This article has not been copyedited and formatted. The final version may differ from this version.
References:
Aksu F, Holmes BB and Fujimoto JM (1993) Opioid antagonists: Indirect antagonism of
morphine analgesia by spinal dynorphin A. Pharmacol Biochem Behav 45:409-418.
Arts KS, Fujimoto JM and Tseng LF (1992) Involvement of dynorphin A and not substance P
in the spinal antianalgesia action of capsaicin against morphine-induced antinociception in
mice. J Pharamcol Exp Ther 261:643-651.
D’Amour FE and Smith DL (1941) A method for determining loss of pain sensation. J Downloaded from
Pharmacol Exp Ther 72:74-79.
Fujimoto JM, Arts KS, Rady JJ and Tseng LF (1990) Spinal dynorphin A (1-17): possible jpet.aspetjournals.org mediator of antianalgesic action. Neuropharmacol 29:609-617.
Gardell LR, Burgess SE, Dogrul A, Ossipov MH, Malan TP, Lai J, Porreca F (2002)
Pronociceptive effects of spinal dynorphin promote cannabinoid-induced pain and at ASPET Journals on September 26, 2021
antinociceptive tolerance. Pain 98: 79-88.
Goldberg IE, Rossi GC, Letchworth SR, Mathis JP, Ryan-Moro J, Leventhal L, Su W, Emmel
D, Bolan EA and Pasternak GW (1998) Pharmacological characterization of endomorphin-
1 and endomorphin-2 in mouse brain. J Pharmacol Exp Ther 286:1007-1013.
Holmes BB and Fujimoto JM (1992) Naloxone and norbinaltorphimine administered
intracerebroventricularly antagonize spinal morphine-induced antinociception in mice
through the antianalgesic action of spinal dynorphin A (1-17). J Pharmacol Exp Ther
261:146-152.
Holmes BB, Fujimoto JM (1993) Inhibiting a spinal dynorphin A component enhances
intrathecal morphine antinociception in mice. Anesth Analg 77:1166-1173.
22 JPET Fast Forward. Published on October 13, 2003 as DOI: 10.1124/jpet.103.056242 This article has not been copyedited and formatted. The final version may differ from this version.
Hung K, Wu H, Mizoguchi H, Tseng LF (2002) Acute antinociceptive tolerance and
unidirectional cross-tolerance to endomorphin-1 and endomorphin-2 given
intraventricularly in the rat. Eur J Pharmacol 448:169-174.
Hylden JLK and Wilcox GL (1980) Intrathecal morphine in mice: a new technique. Eur J
Pharmacol 167:313-316.
Laughlin TM, Vanderah TW, Lashbrook J, Nichols ML, Ossipov M, Porreca F and
Wilcox GL (1997) Spinally administered dynorphin A produces long-lasting Downloaded from
allodynia: involvement of NMDA but not opioid receptors. Pain 72:253-260.
Leitermann RJ, Terashvili M, Mizoguchi H, Chen F, Clithero A and Tseng LF (2003) jpet.aspetjournals.org Release of immunoreactive dynorphin A(1-17) from the spinal cord by intrathecal
perfusion of endomorphin-2, but not endomorphin-1 in anesthetized rats. Soc
Neurosci Abstr 33: 908.12. at ASPET Journals on September 26, 2021
McNally GP (1999) Pain facilitatory circuits in the mammalian central nervous system:
their behavioral significance and role in morphine analgesic tolerance. Neurosci
Biobehav Rev 23:1059-1078.
Milan MJ (1999) The induction of pain: An integrative review. Prog Neurobiol 57:1-164.
Millan MJ (2002) Descending control of pain. Prog Neurobiol 66: 355-474.
Mizoguchi H, Narita M, Nagase H, Tseng LF (1995) Antisense oligodeoxynucleotide to a δ-
opioid receptor blocks the antinociception induced by cold water swimming. Regul Pept
59: 255-259.
Monory K, Bourin M-C, Spetea M, Tomooly C, Toth G, Matthes HW, Kieffer BL, Hanoune J
and Borsodi A (2000) Specific activation of the µ opioid receptor (MOR) by endomorphin
1 and endomorphin 2. Eur J Neuroscience 22: 577-584.
23 JPET Fast Forward. Published on October 13, 2003 as DOI: 10.1124/jpet.103.056242 This article has not been copyedited and formatted. The final version may differ from this version.
Narita M, Mizoguchi H, Oji GS, Tseng EL, Suganuma C, Nagase H and Tseng LF (1998)
Characteriation of endomorphin-1 and –2 on [35S]GTPγS binding in the mouse spinal cord.
Eur J Pharmacol 351:383-387.
Narita M, Mizoguchi H, Narita M, Dun NJ, Hwang BH, Endoh T, Suzuki T, Nagase H,
Suzuki T and Tseng LF (2000) G protein activation by endomorphins in the mouse
periaqueductal gray matter. J Biomed Sci 7:221-225.
Ohsawa M, Mizoguchi H, Narita M, Nagase H, Kampine JP and Tseng LF (2001) Downloaded from
Different antinociception induced by spinally administered endomorphin-1 and
endomorphin-2 in the mouse. J Pharmacol Exp Ther 298:592-597. jpet.aspetjournals.org Pasternak GW (1993) Pharmacological mechanisms of opioid analgesics. Clin
Neuropharmacology 16: 1-18.
Pick CG, Paul D, Pasternak GW (1991) Comparison of naloxonazine and beta- at ASPET Journals on September 26, 2021
funaltrexamine antagonism of µ1 and µ2 opioid actions. Life Sci 48: 2005-2011.
Rady JJ, Fujimoto JM, Tseng LF (1991) Dynorphins other than dynorphin A(1-17) lack
spinal antianalgesic activity but do act on dynorphin A(1-17) receptors. J Pharmacol
Exp Ther 259:1073-1080.
Sakurada S, Hayashi T, Yuhki M, Fujimura T, Murayama K, Yonezawa A, Sakurada C,
Takeshita M, Zadina JE, Kastin AJ and Sakurada T (2000) Different antagonism of
endomorphin-1 and endomorphin-2 spinal antinociception by naloxonazine and 3-
methoxynaltrexone. Brain Res 881:1-8.
Sakurada S, Hayashi T, Yuhki M, Orito T, Zadina JE, Kastin AJ, Fujimura T, Murayama
K, Sakurada C, Sakurada T, Narita M, Suzuki T, Tan-no K, Tseng LF (2001)
24 JPET Fast Forward. Published on October 13, 2003 as DOI: 10.1124/jpet.103.056242 This article has not been copyedited and formatted. The final version may differ from this version.
Differential antinociceptive effects induced by intrathecally administered
endomorphin-1 and endomorphin-2 in the mouse. Eur J Pharmacol 427: 203-210.
Singh L, Oles RJ, Field MJ, Atwal P, Woodruff GN and Hunter JC (1996) Effect of CCK
receptor antagonists on the antinociceptive, reinforcing and gut motility properties of
morphine Br J Pharmacol 118:1317-1325.
Tang Q, Lynch RM, Porreca F and Lai J (2000) Dynorphin A elicits an increase in
intracellular calcium in cultured neurons via a non-opioid, non-NMDA mechanism. J Downloaded from
Neurophysiol 83:2610-2615.
Tseng LF and Collin KA (1993) Spinal involvement of both dynorphin A and met- jpet.aspetjournals.org enkephalin in the antinociception induced by intracerebroventricularly administered
bremazocine but not morphine in the mouse. J Pharmacol Exp Ther 266:1430-1438.
Tseng LF and Huang FY (1992) Release of immunoreactive [Met5]enkephalin and at ASPET Journals on September 26, 2021
cholecystokinin from the spinal cord by β-endorphin administered intraventricularly
in the pentobarbital-anesthetized rat. Eur J Pharmacol 215:309-312.
Tseng LF, Narita M, Suganuma C, Mizoguchi H, Ohsawa M, Nagase H and Kampine JP
(2000) Differential antinociceptive effects of endomorphin-1 and endomorphin-2 in the
mouse. J Pharmacol Exp Ther 292:576-583.
Tseng LF, Narita M, Mizoguchi H, Kawai K, Mizusuna A, Kamei J, Suzuki T, and
Nagase H (1997) Delta-1 opioid receptor-mediated antinociceptive properties of a
nonpeptidic delta opioid receptor agonist, (-) TAN-67, in the mouse spinal cord. J
Pharmacol Exp Ther 280:600-605.
Vanderah TW, Laughlin T, Lashbrook JM, Nichols ML, Wilcox GL, Ossipov MH,
Malan TP and Porreca F (1996) Single intrathecal injections of dynorphin A or des-
25 JPET Fast Forward. Published on October 13, 2003 as DOI: 10.1124/jpet.103.056242 This article has not been copyedited and formatted. The final version may differ from this version.
Tyr-dynorphins produce long-lasting allodynia in rats: blockade by MK-801 but not
naloxone. Pain 68:275-281.
Wang Z, Gardell LR, Ossipov HM, Vanderah TW, Brennan MB, Hochgeschwender U,
Hruby VJ, Malan TP Jr, Lai J, Porreca F (2001) Pronociceptive actions of dynorphin
maintain chronic neuropathic pain. J Neurosci 21: 1779-1786.
Wu H, Hung K, Mizoguchi H, Fujimoto JM, and Tseng LF (2001) Acute antinociceptive
tolerance and asymmetric cross-tolerance between endomorphin-1 and endomorphin- Downloaded from
2 given intracerebroventricularly in the mouse. J Pharmacol Exp Ther 299:1120-
1125. jpet.aspetjournals.org Wu H, Hung K, Mizoguchi H, Nagase H, and Tseng LF (2002a) Roles of endogenous
opioid peptides in modulation of nocifensive response to formalin. J Pharmacol Exp
Ther 300:647-654. at ASPET Journals on September 26, 2021
Wu H, Mizoguchi H, Terashvili M, Leitermann RJ, Hung K, Fujimoto JM, and Tseng LF
(2002b) Spinal pretreatment with antisense oligodeoxynucleotides against exon-1, -4,
or -8 of µ-opioid receptor clone leads to differential loss of spinal endomorphin-1-
and endomorphin-2-induced antinociception in the mouse. J Pharmacol Exp Ther
303:867-873.
Xu JY, and Tseng LF (1997) [Met]enkephalin in the spinal cord is involved in the
antinociception induced by intracerebroventricularly-administered etorphine in the mouse.
Neuroscience 80: 579-585.
Zadina JE, Hackler L, Ge L-J and Kastin AJ (1997) A potent and selective endogenous agonist
for the µ-opioid receptor. Nature 386:499-502.
26 JPET Fast Forward. Published on October 13, 2003 as DOI: 10.1124/jpet.103.056242 This article has not been copyedited and formatted. The final version may differ from this version.
Footnotes: a) This work was supported by Grant DA 03811 from the National Institute of Health,
National Institute on Drug Abuse (PI: L.F.T.). A preliminary report of these results will be
presented at the 33rd Annual Meeting of the Society for Neuroscience, New Orleans, LA,
November 8-12, 2003. b) Leon F. Tseng, Ph.D.
Department of Anesthesiology Downloaded from
Medical Educational Building, M4308
Medical College of Wisconsin jpet.aspetjournals.org 8701 Watertown Plank Road
Milwaukee, WI 53226
USA at ASPET Journals on September 26, 2021
27 JPET Fast Forward. Published on October 13, 2003 as DOI: 10.1124/jpet.103.056242 This article has not been copyedited and formatted. The final version may differ from this version.
Legends for figures:
Fig. 1 The TF responses to i.t. administration of EM-1, EM-2 or vehicle. Groups of mice were injected i.t. with EM-1 (1.64 nmol), EM-2 (1.75 nmol), or vehicle (5 µl) and the TF responses were then measured at different times after injection. Each point represents the mean and vertical bar represents the S.E.M. of %MPE with 8 mice in each group. Two- way ANOVA followed by Bonferroni post-test was used to test the difference between groups. For group of mice injected with EM-1 vs. vehicle, F interaction, treatment, time = 3.06, Downloaded from
14.52, 7.28 and EM-2 vs vehicle, F interaction, treatment, time = 1.14, 12.08, 3.70; * p < 0.001.
jpet.aspetjournals.org Fig. 2 The TF responses to i.t. administration of morphine in mice pretreated i.t. with
EM-1, EM-2 or vehicle. Groups of mice were injected i.t. with EM-1 (1.64 nmol), EM-2
(1.75 nmol), or vehicle 45 min prior to i.t. injection of morphine (3.0 nmol) and the TF at ASPET Journals on September 26, 2021 responses were measured at various times after injection. Each point represents the mean and vertical bar represents the S.E.M. with 8 to 10 mice in each group. Two-way
ANOVA followed by Bonferroni post-test was used to test the difference between groups. For group of mice injected with EM-2 vs vehicle injected mice, F interaction, treatment, time = 15.17, 102.3, 32.91 and EM-1 vs. vehicle injected mice, F interaction, treatment, time =
1.97, 1.79, 43.21; *p < 0.001.
Fig. 3 Effect of different pretreatment times with EM-2 (A) and different doses of EM-2
(B) given i.t. on the TF inhibition induced by i.t.-administered morphine. (A) Groups of mice were pretreated i.t. (5 µl each) with EM-2 (1.75 nmol) or vehicle 15, 30, 45, 60, 90, or 120 min before i.t. administration of morphine (3.0 nmol). (B) Groups of mice were
28 JPET Fast Forward. Published on October 13, 2003 as DOI: 10.1124/jpet.103.056242 This article has not been copyedited and formatted. The final version may differ from this version.
pretreated i.t. with EM-2 (0.02 to 17.5 nmol) or vehicle 45 min prior to i.t. injection of morphine (3.0 nmol). The TF response (% MPE) was measured 15 min after morphine administration. Each column represents the mean and the vertical bar represents the
S.E.M. with 8 to 13 mice in each group. Two-way ANOVA followed by Bonferroni's post-test (A) and one-way ANOVA followed by Dunnett’s post-test (B) were used to test the difference between groups. For group of mice injected with 1.75 nmol of EM-2 vs.
vehicle injected mice, F interaction, treatment, time = 6.14, 25.62, 6.92, * p < 0.01 and ** p < Downloaded from
0.001(figure 3A). For groups of mice pretreated with different doses of vs. vehicle injected mice, F = 12.70, ** p < 0.001 (Figure 3B). jpet.aspetjournals.org
Fig. 4 Effect of i.t. pretreatment with naloxone on i.t. morphine-induced TF inhibition in mice also pretreated i.t. with EM-2. Groups of mice were pretreated i.t. with naloxone at ASPET Journals on September 26, 2021
(5.5, 16.5, 55 pmol) or vehicle 10 min before i.t. injection of EM-2 (1.75 nmol) and injected i.t. with morphine (3.0 nmol) 45 min after EM-2 injection. One group of mice were i.t. injected with morphine (3.0 nmol) alone as negative control. The TF inhibition
(% MPE) was measured 15 min after morphine injection. Each column represents the mean and the vertical bar represents the S.E.M. with 8 to 11 mice in each group. The one-way ANOVA followed by Dunnett’s post-test was used to test the difference between groups. For groups of mice pretreated with different doses of EM-2 vs. vehicle pretreated mice, F = 12.70, * p < 0.001.
Fig. 5 Effects of i.t. pretreatment with 3-methoxynaltexone, naltrindole(NTI), nor-BNI or MK801 on i.t. morphine-induced TF inhibition in mice also pretreated i.t. with EM-2.
29 JPET Fast Forward. Published on October 13, 2003 as DOI: 10.1124/jpet.103.056242 This article has not been copyedited and formatted. The final version may differ from this version.
Groups of mice were i.t. pretreated with µ-opioid receptor antagonist 3- methoxynaltrexone 6.4 pmol 25 min, δ-opioid receptor antagonist NTI 11.1 nmol 10 min,
κ-opioid receptor antagonist nor-BNI 6.6 nmol 24 h, or NMDA receptor antagonist MK-
801 10 nmol 20 min before i.t. injection of EM-2 (1.75 nmol) and were then injected i.t. with morphine (3.0 nmol) 45 min after EM-2 injection. One group of mice were i.t. injected with morphine (3.0 nmol) alone as negative control. The TF response (% MPE) was measured 15 min after morphine administration. Each column represents the mean Downloaded from and the vertical bar represents the S.E.M. with 8 to 10 mice in each group. The Student t- test was used to test the difference between groups. * p < 0.001 compared with vehicle- jpet.aspetjournals.org injected groups.
Fig. 6 Effect of i.t.pretreatment with various doses of antiserum against dynorphin A(1- at ASPET Journals on September 26, 2021
17) on the i.t. morphine-induced TF inhibition in mice also pretreated i.t. with EM-2.
Groups of mice were pretreated i.t. with antiserum against Dyn or NRS (50, 100, 200,
300 µg) 1 h before i.t. injection with EM-2 (1.75 nmol) and then injected i.t. with morphine (3.0 nmol) 45 min after EM-2 injection. One group of mice were i.t. injected with morphine (3.0 nmol) alone as negative control. The TF inhibition (% MPE) was measured 15 min after morphine administration. Each column represents the mean and the vertical bar represents the S.E.M. with 9 to 10 mice in each group. Two-way
ANOVA followed by Bonferroni post-test were used to test the difference between groups. For groups of mice injected with antiserum against Dyn vs NRS, F interaction, treatment, time = 2.96, 37.65, 8.57; * p < 0.01, ** p < 0.001 compared with NRS-injected groups.
30 JPET Fast Forward. Published on October 13, 2003 as DOI: 10.1124/jpet.103.056242 This article has not been copyedited and formatted. The final version may differ from this version.
Fig. 7 Effect of i.t. pretreament with EM-2 on the TF inhibition induced by i.t. administration of opioid µ, δ and κ agonists. Groups of mice were pretreated i.t. with
EM-2 (1.75 nmol) or vehicle 45 min before i.t. injection with DAMGO (19.5 pmol), EM-
1 (16.4 nmol), EM-2 (35 nmol), deltorphin II (6.4 nmol), or U50,488H (123.2 nmol). TF inhibition was measured 5 min after EM-1 or EM-2 and 10 min after DAMGO, deltorphin II or U50,488H injection. Each column represents the mean and the vertical Downloaded from bar represents the S.E.M. with 7 to 10 mice in each group. Student t-test was used to test the difference. * p < 0.001 compared with vehicle-injected groups. jpet.aspetjournals.org at ASPET Journals on September 26, 2021
31 JPET Fast Forward. Published on October 13, 2003 as DOI: 10.1124/jpet.103.056242 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021 JPET Fast Forward. Published on October 13, 2003 as DOI: 10.1124/jpet.103.056242 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021 JPET Fast Forward. Published on October 13, 2003 as DOI: 10.1124/jpet.103.056242 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021 JPET Fast Forward. Published on October 13, 2003 as DOI: 10.1124/jpet.103.056242 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021 JPET Fast Forward. Published on October 13, 2003 as DOI: 10.1124/jpet.103.056242 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021 JPET Fast Forward. Published on October 13, 2003 as DOI: 10.1124/jpet.103.056242 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021 JPET Fast Forward. Published on October 13, 2003 as DOI: 10.1124/jpet.103.056242 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021 JPET Fast Forward. Published on October 13, 2003 as DOI: 10.1124/jpet.103.056242 This article has not been copyedited and formatted. The final version may differ from this version.
Table 1. Effects of i.t. pretreatment with antiserum against dynorphin A(1-17),
[Met]-enkephalin, [Leu]-enkepahlin, β-endorphin or cholecystokinin on the tail-flick inhibition induced by i.t.-administered morphine in mice pretreated i.t. with EM-2.
Groups of mice were injected i.t. with the antiserum (200 µg each) against dynorphin
A(1-17), [Met]-enkephalin, [Leu]-enkepahlin, β-endorphin, cholecystokinin or normal rabbit serum 1 h prior to i.t. injection of EM-2 (1.75 nmol) and were then injected i.t. with morphine (3.0 nmol) 45 min after EM-2 injection. The tail-flick response was Downloaded from measured 15 min after morphine injection.
Pretreatment Antinociception (%MPE)a jpet.aspetjournals.org
Normal rabbit serum 34.27 ± 5.18 (8)
A/S Dynorphin A(1-17) 73.19 ± 9.39b (9)
A/S [Met]enkephalin 26.92 ± 6.01 (8) at ASPET Journals on September 26, 2021
A/S [Leu]enkephalin 24.63 ± 9.39 (8)
A/S β-endorphin 25.30 ± 10.0 (9)
A/S Cholescystokinin 36.91 ± 5.30 (8) a Data were expressed as mean ± S.E. Number in parenthesis denote the number of mice studied. b One-way ANOVA (F = 5.64) followed by Dunnett’s post-test; *p < 0.001, compared with mice injected with normal rabbit serum.