d-Pro2-Endomorphin-1 andd-Pro2-Endomorphin-2, Respectively, Attenuate the Antinociception Induced by Endomorphin-1 and Endomorphin-2 Given Intrathecally in the Mouse

  1. Kuei-chun Hung1,
  2. Hsiang-en Wu1,
  3. Hirokazu Mizoguchi1,
  4. Shinobu Sakurada2,
  5. Toru Okayama3,
  6. Tsutomu Fujimura4,
  7. Kimie Murayama4,
  8. Tsukasa Sakurada5,
  9. James M. Fujimoto1 and
  10. Leon F. Tseng1
  1. 1Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin (K.-C.H., H.-E.W., H.M., J.M.F., L.F.T.);2Department of Physiology and Anatomy, Tohoku Pharmaceutical University, Sendai, Japan (S.S.); 3Research Institute, Fuji Chemical Industries Ltd., Takaoka, Japan (T.O.); 4Central Laboratory of Medical Sciences, Juntendo University School of Medicine, Tokyo, Japan (T.F., K.M.); and5Department of Biochemistry, Daiichi College of Pharmaceutical Science, Fukuoka, Japan (T.S.)
  1. Dr. Leon F. Tseng, Department of Anesthesiology, Medical Education Bldg., Room M4308, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. E-mail: ltseng{at}mcw.edu
 
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Abstract

First, the antinociception with the tail-flick test ofd-Pro2-endomorphin-1 andd-Pro2-endomorphin-2 given i.t. was compared with that produced by endomorphin-1 and -2 in male CD-1 mice. High doses of d-Pro2-endomorphin-1 (0.2–0.4 pmol) and d-Pro2-endomorphin-2 (300–800 pmol) given i.t. produced antinociception with low intrinsic activity [about 25% maximum possible effect (MPE)] compared with that of endomorphin-1 (16.4 nmol) and endomorphin-2 (35 nmol) (>90% MPE). Second, coadministration of a low dose ofd-Pro2-endomorphin-1 (0.1 pmol), which given alone did not affect the tail-flick latencies, markedly attenuated the antinociception induced by endomorphin-1 (16.4 nmol) but not by endomorphin-2 (35 nmol). Similarly, coadministration of a low dose ofd-Pro2-endomorphin-2 (200 pmol), which given alone did not affect the tail-flick latencies, significantly attenuated the antinociception induced by endomorphin-2 (35 nmol) and, to a much lesser extent, endomorphin-1 (16.4 nmol). It is concluded thatd-Pro2-endomorphin-1 andd-Pro2-endomorphin-2 at high doses were partial opioid receptor agonists to produce antinociception, and at low doses were opioid receptor antagonists to block selectively the antinociception induced by endomorphin-1 and endomorphin-2, respectively. Furthermore, our results are consistent with the view that the antinociception induced by endomorphin-1 and endomorphin-2 is mediated by the stimulation of different subtypes of μ-opioid receptors.

Endomorphin-1 and endomorphin-2 are two endogenous tetrapeptides isolated from the bovine frontal cortex (Zadina et al., 1997) and human brain (Hackler et al., 1997). These peptides are the first endogenous peptides to be proposed to have high affinity and selectivity for μ-opioid receptors. Receptor-binding assays and immunocytochemical studies reveal that endomorphin-1 and endomorphin-2 potently compete with μ1- and μ2-receptors and that they are widely located at the sites in the brain and spinal cord abundant in μ-opioid receptors (Martin-Schild et al., 1997,1998, 1999; Goldberg et al., 1998; Pierce et al., 1998; Shreff et al., 1998; Wu et al., 1999). Through the stimulation of μ-opioid receptors, endomorphin-1 and endomorphin-2 inhibit the electrical activity of rostral ventrolateral medulla neurons or spinal substantia gelatinosa neurons (Chu et al., 1999; Wu et al., 1999). The release of endomorphin-2 from the spinal cord can be achieved by electrical stimulation (Williams et al., 1999). The administration of endomorphin-1 and endomorphin-2 given i.c.v. and i.t. produces potent antinociceptive responses that are blocked by μ-opioid receptors antagonists naloxone, β-funaltrexamine, andd-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH2(CTOP) (Zadina et al., 1997; Stone et al., 1997; Narita et al., 1998;Tseng et al., 2000). Neither endomorphin-1 nor endomorphin-2 activates μ-opioid receptor-coupled G proteins in μ-opioid receptor knockout mice, and the antinociception induced by endomorphin-1 and endomorphin-2 is attenuated in heterozygous knockout mice and virtually abolished in homozygous knockout mice (Mizoguchi et al., 1999). These findings support the view that antinociception induced by the endomorphin-1 and endomorphin-2 is mediated by the stimulation of μ-opioid receptors.

However, more recent results illustrate that different subtypes of μ-opioid receptors may be involved in antinociceptive effects induced by endomorphin-1 and endomorphin-2. For example, Sakurada et al. (1999)reported that μ1-opioid receptor antagonist naloxonazine was more effective in blocking the antinociceptive effects induced by endomorphin-2 than endomorphin-1 in mice. The antinociception induced by endomorphin-1 is blocked by μ-opioid receptor antagonists CTOP or β-funaltrexamine (β-FNA) but not by κ-opioid antagonist nor-binaltorphimine (nor-BNI). On the other hand, the antinociception induced by endomorphin-2 is blocked by CTOP or β-FNA and also by nor-BNI (Tseng et al., 2000; Ohsawa et al., 2001). The findings are taken to indicate that two different subtypes of μ-opioid receptors are involved in endomorphin-1- and endomorphin-2-induced antinociception.

d-Pro2-Endomorphins, analogs of endomorphins containing d-amino acid isomers, were designed to examine whether these analogs produce antinociception after i.t. administration. Also, the ability of the analogs to modify antinociception induced by i.t. endomorphin-1 and endomorphin-2 was investigated.

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Materials and Methods

Animals.

Male CD-1 mice (Charles River Laboratories, Inc., Wilmington, MA), weighing 25 to 30 g were used. Animals were housed five per group in a room maintained at 22 ± 0.5°C with an alternating 12-h light/dark cycle. Food and water were available ad libitum.

Drugs.

Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) were purchased from Calbiochem (La Jolla, CA). The peptides were dissolved in sterile saline solution (0.9% NaCl solution) containing 10% hydroxypropyl-β-cyclodextrin for i.t. injection.d-Pro2-Endomorphin-1 (Tyr-d-Pro-Trp-Phe-NH2) andd-Pro2-endomorphin-2 (Tyr-d-Pro-Phe-Phe-NH2) were obtained from Dr. T. Sakurada (Department of Biochemistry, Daiichi College of Pharmaceutical Science, Fukuoka, Japan). Thesed-Pro2-endomorphins were first completely dissolved in dimethyl sulfoxide and then 0.9% sodium chloride solution was added to a final concentration of dimethyl sulfoxide at 1%.

Assessment of Antinociceptive Response.

The antinociceptive response was assessed with the thermal tail-flick test (D'Amour and Smith, 1941). Mice were gently held with the tail positioned in the tail-flick apparatus (model TF6; EMDIE Instrument Co., Maidens, VA) for radiant heat stimulation of the dorsal surface of the tail. The intensity of the heat stimulus was adjusted to cause the animal to flick its tail within 3 to 4 s as the baseline of latency. After measuring the latency, different groups of mice were treated with endomorphin-1, endomorphin-2, or vehicle given i.t., and the tail-flick responses were then measured at different times after injection. The data were expressed as percentage of maximum possible effect (%MPE), which was calculated as [T1 −T0)/(T2−T0)] × 100.T0 and T1 were predrug and postdrug latency, respectively, andT2 was the cutoff time that was set at 10 s to minimize tissue damage.

Drug Administration Protocol.

The i.t. injection (5 μl) was performed according to the procedure of Hylden and Wilcox (1980)using a 25-μl Hamilton syringe with a 30-gauge needle. Groups of mice were treated i.t. with various doses of endomorphin-1 (0.82–16.4 nmol), endomorphin-2 (1.75–35 nmol),d-Pro2-endomorphin-1 (0.03–0.4 pmol), d-Pro2-endomorphin-2 (50–800 pmol), or vehicle, and the tail-flick tests were performed at 2.5, 5, 7.5, 10, 15, and 20 min thereafter. The effects ofd-Pro2-endomorphin-1 andd-Pro2-endomorphin-2 on the tail-flick inhibition induced by endomorphin-1 and endomorphin-2 were studied. Groups of mice were coadministered i.t. with various doses ofd-Pro2-endomorphin-1 (0.03–0.4 pmol) or d-Pro2-endomorphin-2 (50–800 pmol) with endomorphin-1 (16.4 nmol) or endomorphin-2 (35 nmol) and the tail-flick response was measured thereafter. In another experiment, groups of mice were coadministered i.t. withd-Pro2-endomorphin-1 (0.1 pmol) ord-Pro2-endomorphin-2 (200 pmol) with various doses of endomorphin-1 (0.82–16.4 nmol) or endomorphin-2 (1.75–35 nmol), and the tail-flick response was measured thereafter. To establish dose-response curves, at least four doses were used with 8 to 11 mice at each dose. For the calculation of the ED50 values for endomorphin-1- and endomorphin-2-induced tail-flick inhibition, the antinociception was assessed using peak effect, which occurred at either 2.5 or 5 min after administration.

Statistical Analysis.

The data were expressed as the mean with S.E.M. The maximal %MPE was used to graph dose-response curves for endomorphin-1 and endomorphin-2. GraphPad Prism software (version 3.0; GraphPad Software, San Diego, CA) was used to calculate dose-response curves, ED50 values and their confidence intervals. A two-way ANOVA followed by Bonferroni's post test was used to determine the time in which the attenuation of antinociception reached a maximum byd-Pro2-endomorphin-1 ord-Pro2-endomorphin-2 administration. A one-way ANOVA followed by Dunnett's test was used to compare the difference for each group versus the control group.

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Results

Time Courses and Dose Effects of i.t. Administration of Endomorphin-1, Endomorphin-2,d-Pro2-Endomorphin-1, andd-Pro2-Endomorphin-2 on Tail-Flick Response.

Groups of mice were injected i.t. with different doses of endomorphin-1, endomorphin-2,d-Pro2-endomorphin-1,d-Pro2-endomorphin-2, or vehicle, and the tail-flick response was measured 2.5, 5, 7.5, 10, 15, and 20 min after injection. Intrathecal injection of endomorphin-1 at doses 0.82 to 16.4 nmol (Fig. 1A) and endomorphin-2 at doses 1.75 to 35.0 nmol (Fig. 1B) dose- and time-dependently produced inhibition of the tail-flick response. The inhibition of the tail-flick response induced by endomorphin-1 and endomorphin-2 developed rapidly, reached their peak at 5 min, declined rapidly, and returned to the preinjection level in 20 min. A dose 16.4 nmol of endomorphin-1 or 35 nmol of endomorphin-2 produced about 90% MPE. The antinociceptive ED50 values of endomorphin-1 and endomorphin-2 are shown in Table 1. Endomorphin-1 was found to be 2.3-fold more potent than endomorphin-2 to produce the tail-flick inhibition.

Figure 1
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Figure 1

Time course of changes in the tail-flick inhibition induced by i.t.-injected endomorphin-1 (EM-1; A) and endomorphin-2 (EM-2; B). Groups of mice were injected i.t. with different doses of endomorphin-1, endomorphin-2, or vehicle (5 μl), and tail-flick responses were measured at different times after injection. Each value represents the mean ± S.E.M. with 8 to 12 mice/group.

View this table:
Table 1

The antinociceptive potencies (ED50) and the slope function of dose-response curves for i.t. administration of endomorphin-1 and endomorphin-2 given alone or withd-Pro2-endomorphin-1 ord-Pro2-endomorphin-2

d-Pro2-Endomorphin-1 (0.1–0.4 pmol) (Fig. 2A) andd-Pro2-endomorphin-2 (200–800 pmol) (Fig. 2B) given i.t. produced an apparent dose-dependent inhibition of the tail-flick response. However, at the three highest doses of each, there was a ceiling effect (about 25% MPE) where the increase in dose did not lead to a greater effect. It seemed thatd-Pro2-endomorphin-1 was about 2000-fold more potent thand-Pro2-endomorphin-2 to produce the same ceiling effect. A small dose ofd-Pro2-endomorphin-1 (0.1 pmol) ord-Pro2-endomorphin-2 (200 pmol) did not show any appreciable tail-flick inhibition.

Figure 2
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Figure 2

Tail-flick response after i.t. administration of d-Pro2-endomorphin-1 (d-Pro-EM-1; A) ord-Pro2-endomorphin-2 (d-Pro-EM-2; B). Groups of mice were injected i.t. with various doses ofd-Pro2-endomorphin-1 (0.03–0.4 pmol),d-Pro2-endomorphin-2 (50–800 pmol), or vehicle, and tail-flick responses were measured 2.5, 5, 7.5, 10, 15, and 20 min after administration. Administration ofd-Pro2-endomorphin-1 andd-Pro2-endomorphin-2 dose dependently increased the tail-flick latencies, reached the peaks either 2.5 or 5 min, and declined to the preinjection levels in 20 min after injection. The peak tail-flick inhibition of each mouse, which was occurred either at 2.5 or 5 min after injection, was used to assess the antinociceptive effects of d-Pro2-endomorphin-1 andd-Pro2-endomorphin-2. Each column represents the mean ± S.E.M. with 8 to 12 mice/group. A one-way ANOVA followed by Dunnett's test was used to compare the difference for each group versus the vehicle group. ★★★, P < 0.001.

Effects of d-Pro2-Endomorphin-1 andd-Pro2-Endomorphin-2 on Tail-Flick Inhibition Induced by Endomorphin-1 and Endomorphin-2, Respectively.

The presence of low, ceiling antinociceptive action suggested that these analogs might be acting on the same respective opioid receptors as were endomorphin-1 and endomorphin-2. Then, it might be possible that either a positive or negative effect might be shown for these analogs to modify antinociception produced by endomorphin-1 and endomorphin-2. Groups of mice were administered with various doses ofd-Pro2-endomorphin-1 (0.03–0.4 pmol) together with an antinociceptive dose of 16.4 nmol of endomorphin-1. Treatment withd-Pro2-endomorphin-1 at 0.1 pmol, but not at other higher or lower doses significantly attenuated the tail-flick inhibition induced by endomorphin-1 (Fig.3A).

Figure 3
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Figure 3

Effects of i.t. treatment ofd-Pro2-endomorphin-1 andd-Pro2-endomorphin-2 on the inhibition of the tail-flick response induced by endomorphin-1 (A) and endomorphin-2 (B). Groups of mice were treated with different doses ofd-Pro2-endomorphin-1 ord-Pro2-endomorphin-2 mixed with endomorphin-1 with a final dose of 16.4 nmol or endomorphin-2 with a final dose of 35 nmol. The tail-flick response was measured after the injection. Each column represents the mean ± S.E.M. with 8 to 12 mice/group. A one-way ANOVA followed by Dunnett's test was used to compare the difference for each group versus the control group. ★,P < 0.05; ★★★, P < 0.001 compared with the mice treated with endomorphin alone.

A similar experiment was performed for the interaction ofd-Pro2-endomorphin-2 with endomorphin-2. d-Pro2-Endomorphin-2 at 100, 200, and 300 pmol attenuated the tail-flick inhibition induced by endomorphin-2 (35 nmol) (Fig. 3B).

A crossover experiment then performed with the effective doses of the analogs. As seen in Fig. 3A, 200 pmol ofd-Pro2-endomorphin-2 had a significant effect to reduce the tail-flick inhibition induced by endomorphin-1. However, the effect did not seem to be as great as it was against endomorphin-2 (Fig. 3B). Whend-Pro2-endomorphin-1 was evaluated against endomorphin-2-induced tail-flick inhibition, it had no significant effect (Fig. 3B). Thus, the data indicated that there was minimal crossover of the effects ofd-Pro2-endomorphin-1 andd-Pro2-endomorphin-2 relative to their selectivity of action against endomorphin-1- and endomorphin-2-mediated antinociception.

In the preceding set of experiments, the analogs were given at the same time as endomorphin-1 and endomorphin-2. In the experiment given in Fig. 4, A and B, the time of treatment with the analogs was given 0 (together), 5, and 10 min separately before the endomorphin-1 and endomorphin-2, and tail-flick responses were then measured after the injection. In each case, the respective analogs were most effective when each was given at the same time (0 min) as the endomorphin-1 and endomorphin-2.

Figure 4
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Figure 4

Time course of i.t. pretreatment withd-Pro2-endomorphin-1 (A) andd-Pro2-endomorphin-2 (B) on the inhibition of the tail-flick response induced by i.t. injection of endomorphin-1 and endomorphin-2, respectively. A, groups of mice were pretreated with 0.1 pmol of d-Pro2-endomorphin-1 or vehicle (5 μl) 0, 5, or 10 min before i.t. administration of endomorphin-1 (16.4 nmol). Tail-flick responses were then measured after each injection. B, groups of mice were pretreated with 200 pmol ofd-Pro2-endomorphin-2 or vehicle (5 μl) 0, 5, or 10 min before i.t. administration of endomorphin-2 (35 nmol), and tail-flick responses were measured after the injection. Each column represents the mean, and the vertical bar represents the S.E.M. with 8 to 11 mice/group. A two-way ANOVA followed by Bonferroni's post test was used to test the difference among groups. ★★,P < 0.01; ★★★, P < 0.001 compared with mice pretreated with the vehicle.

Effects of i.t. Treatment with a Fixed Dose ofd-Pro2-Endomorphin-1 andd-Pro2-Endomorphin-2 on Dose-Response Curves for i.t. Endomorphin-1- and Endomorphin-2-Induced Tail-Flick Inhibition.

Endomorphin-1 at doses 0.82 to16.4 nmol or endomorphin-2 at doses 1.75 to 35 nmol given i.t. dose-dependently inhibited the tail-flick response. The ED50values, Hill slope function, and their 95% confidence intervals are given in Table 1. Intrathecal coadministration of 0.1 pmol ofd-Pro2-endomorphin-1 with endomorphin-1 markedly attenuated the antinociceptive response induced by endomorphin-1; the dose-response curve for endomorphin-1 was shifted to the right by 5.5-fold. The shift of the dose-response curve for endomorphin-1-induced tail-flick inhibition afterd-Pro2-endomorphin-1 was not significantly deviated from parallel (Fig.5A; Table 1). Similarly, i.t. coadministration of 200 pmol ofd-Pro2-endomorphin-2 with endomorphin-2 markedly attenuated the antinociceptive response induced by endomorphin-2; the dose-response curve for endomorphin-2 was shifted to the right by 3.9-fold. The shift of the dose-response curve for endomorphin-2-induced tail-flick inhibition afterd-Pro2-endomorphin-2 was not significantly deviated from parallel (Fig. 5B; Table 1).

Figure 5
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Figure 5

Effects of i.t. treatment withd-Pro2-endomorphin-1 andd-Pro2-endomorphin-2 on dose-response curves for the inhibition of the tail-flick response induced by i.t.-administered endomorphin-1 (A) and endomorphin-2 (B). Groups of mice were simultaneously treated with various doses of endomorphin-1 and 0.1 pmol of d-Pro2-endomoprhin-1 or various doses of endomorphin-2 and 200 pmol ofd-Pro2-endomorphin-2. Tail-flick responses were measured then after the injection. Each vertical column represents the mean ± S.E.M. with 8 to 12 mice/group.

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Discussion

As in our previous report (Ohsawa et al., 2001), i.t. administration of endomorphin-1 and endomorphin-2 produced antinociception with full intrinsic activity (>90% MPE). Endomorphin-1 was about 2.3- fold more potent than endomoprhin-2. The analogs of endomorphins,d-Pro2-endomorphin-1 andd-Pro2-endomorphin-2, at high doses, also produced antinociception, but with low intrinsic activity (about 25% MPE) and a ceiling effect. Thus,d-Pro2-endomorphin-1 andd-Pro2-endomorphin-2 were partial agonists compared with endomorphin-1 and endomorphin-2. The antinociceptive properties ofd-Pro2-endomorphin-1 andd-Pro2-endomorphin-2 we found in the present studies in mice are consistent with the findings by others in rats (Shane et al., 1999; Krzanowska et al., 2000).

We found in the present studies thatd-Pro2-endomorphin-1 andd-Pro2-endomorphin-2 at small doses are antagonists and antagonized the antinociception induced by endomorphin-1 and endomorphin-2, respectively. Coadministration ofd-Pro2-endomorphin-2 with endomorphin-2 given i.t. attenuated the antinociception induced by endomorphin-2 and only slightly attenuated the antinociception induced by endomorphin-1. These results suggest thatd-Pro2-endomorphin-2 seem to block the μ-opioid receptors predominantly stimulated by endomorphin-2 and to a lesser extent μ-opioid receptors stimulated by endomorphin-1. In addition, coadministration withd-Pro2-endomorphin-1 with endomorphin-1 attenuated the antinociception induced by endomorphin-1, but not endomorphin-2, indicating thatd-Pro2-endomorhin-1 blocks μ-opioid receptors stimulated by endomorphin-1 only but not by endomorphin-2. The results of our present studies provide additional evidence to support the view that the antinociception induced by endomorphin-1 and endomorphin-2 is mediated by the stimulation of different subtypes of μ-opioid receptors.

d-Pro2-Endomorphin-1 andd-Pro2-endomorphin-2 blocked the antinociception induced by endomorphin-1 and endomorphin-2 only at small doses, but not at high doses. Only 0.1 pmol ofd-Pro2-endomorphin and 100 to 300 pmol of d-Pro2-endomorphin-2, but not higher doses, blocked the antinociception induced by endomorphin-1 and endomorphin-2, respectively. Becaused-Pro2-endomorphin-1 andd-Pro2-endomorphin-2 at high doses produced a weak antinociception with low intrinsic activity and ceiling effect, the failure for high doses ofd-Pro2-endomorphin-1 ord-Pro2-endomorphin-2 to block the endomorphin-1- or endomorphin-2-induced antinociception is unexpected. The endomorphin-1 was found to be only 2.3-fold more potent than endomorphin-2 to produce the antinociception, butd-Pro2-endomorphn-1 was found to be at least 2000-fold more potent thand-Pro2-endomorphin-2 to produce the antinociception and to block the antinociception induced by endomorphin-1 and endomorphin-2, respectively (Figs. 2 and 3; Table 1). It is possible thatd-Pro2-endomorphin-1 andd-Pro2-endomorphin-2 may, respectively, bind to different subtypes of μ-opioid receptors to produce agonistic and antagonistic effects.

The view that different subtypes of μ-opioid receptors are involved in endomorphin-1- and endomorphin-2-induced antinociception has been reported (Sakurada et al., 1999, 2000; Ohsawa et al., 2000; Wu et al., 2001). Pretreatment with μ1-opioid receptor antagonist naloxonazine is more effective in antagonizing the antinociception induced by endomorphin-2 than endomorphin-1. Also pretreatment with 3-methynaltrexone, a morphine-6β-glucuronide antagonist, blocks the antinociception induced by endomorphin-2, but not endomorphin-1 (Sakurada et al., 1999, 2000). A unidirectional cross-tolerance between endomorphin-1 and endomorphin-2 in mice has been reported. Mice made tolerant to endomorphin-1 by i.c.v. pretreatment with endomorphin-1 exhibit nearly no cross-tolerance to endomorphin-2 to produce antinociception. On the other hand, mice made tolerant to endomorphin-2 exhibit partial cross-tolerance to endomorphin-1 (Wu et al., 2001). The antinociception induced by endomorphin-1 is blocked by μ-opioid receptor antagonists CTOP or β-FNA but not by κ-opioid antagonist nor-BNI. On the other hand, the antinociception induced by endomorphin-2 is blocked by CTOP, β-FNA, or nor-BNI (Tseng et al., 2000; Ohsawa et al., 2001). Furthermore, i.t. pretreatment with antiserum against dynorphin A(1-17) blocks the antinociception induced by endomorphin-2, but not endomorphin-1. Thus, the antinociception induced by endomorphin-1 and endomoprhin-2 is mediated by the stimulation of different subtypes of μ-opioid receptors. The endomorphin-2-induced antinociception contains an additional component, which is mediated by the spinal release of dynorphin A(1-17) acting on κ-opioid receptors in the spinal cords (Ohsawa et al., 2001).

In opioid receptor binding assay in mouse brain homogenates, both endomorphin-1 and endomorphin-2 compete for both μ1- and μ2-receptor binding sites potently, consistent with the view that the antinociception induced by endomorphin-1 and endomorphin-2 is primarily mediated by the stimulation of μ-opioid receptors. However, the study was unable to identify the presence of different subtypes of μ-opioid receptors for endomorphin-1 and endomorphin-2 (Goldberg et al., 1998).

It is concluded thatd-Pro2-endomorphin-1 andd-Pro2-endomorphin-2 at high doses are partial opioid agonists that produce week antinociception and at low doses are antagonists to block selectively the antinociception induced by endomorphin-1 and endomorphin-2, respectively. Our results provide evidence that the antinociception induced by endomorphin-1 and endomorphin-2 is mediated by the stimulation of different subtypes of μ-opioid receptors.

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Footnotes

  • This work was supported in part by Grant DA 03811 from the National Institute of Health, National Institute on Drug Abuse (to L.F.T.).

  • DOI: 10.1124/jpet.102.038927

  • Abbreviations:
    CTOP
    d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH2
    β-FNA
    β-funaltrexamine
    nor-BNI
    nor-binaltorphimine
    MPE
    maximum possible effect
    ANOVA
    analysis of variance
    d-Pro-EM-1
    d-Pro2-endomorphin-1
    d-Pro-EM-2
    d-Pro2-endomorphin-2
    • Received May 15, 2002.
    • Accepted August 2, 2002.
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  1. doi: 10.1124/jpet.102.038927 JPET November 1, 2002 vol. 303 no. 2 874-879
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