Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

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, -funaltrexamine, and D-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 -funaltrexamine (-FNA) but not by -opioid antagonist nor-binaltorphimine (nor-BNI). On the other hand, the antinociception induced by endomorphin-2 is blocked by CTOP or -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.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

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--cyclodextrin for i.t. injection. D-Pro²-Endomorphin-1 (Tyr-D-Pro-Trp-Phe-NH₂) and D-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). These D-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, and T₂ 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 of D-Pro²-endomorphin-1 and D-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 of D-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. with D-Pro²-endomorphin-1 (0.1 pmol) or D-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 by D-Pro²-endomorphin-1 or D-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.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Time Courses and Dose Effects of i.t. Administration of Endomorphin-1, Endomorphin-2, D-Pro²-Endomorphin-1, and D-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.

View larger version (24K): [in this window] [in a new window]   Fig. 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 larger version (17K): [in this window] [in a new window]   Fig. 2.   Tail-flick response after i.t. administration of D-Pro2-endomorphin-1 (D-Pro-EM-1; A) or D-Pro2-endomorphin-2 (D-Pro-EM-2; B). Groups of mice were injected i.t. with various doses of D-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 of D-Pro2-endomorphin-1 and D-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 and D-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 and D-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 of D-Pro²-endomorphin-1 (0.03-0.4 pmol) together with an antinociceptive dose of 16.4 nmol of endomorphin-1. Treatment with D-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).

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Effects of i.t. treatment of D-Pro2-endomorphin-1 and D-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 of D-Pro2-endomorphin-1 or D-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.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Time course of i.t. pretreatment with D-Pro2-endomorphin-1 (A) and D-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 of D-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 of D-Pro²-Endomorphin-1 and D-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 ED₅₀ values, Hill slope function, and their 95% confidence intervals are given in Table 1. Intrathecal coadministration of 0.1 pmol of D-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 after D-Pro²-endomorphin-1 was not significantly deviated from parallel (Fig. 5A; Table 1). Similarly, i.t. coadministration of 200 pmol of D-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 after D-Pro²-endomorphin-2 was not significantly deviated from parallel (Fig. 5B; Table 1).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Effects of i.t. treatment with D-Pro2-endomorphin-1 and D-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 of D-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.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

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 and D-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 and D-Pro²-endomorphin-2 were partial agonists compared with endomorphin-1 and endomorphin-2. The antinociceptive properties of D-Pro²-endomorphin-1 and D-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 that D-Pro²-endomorphin-1 and D-Pro²-endomorphin-2 at small doses are antagonists and antagonized the antinociception induced by endomorphin-1 and endomorphin-2, respectively. Coadministration of D-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 that D-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 with D-Pro²-endomorphin-1 with endomorphin-1 attenuated the antinociception induced by endomorphin-1, but not endomorphin-2, indicating that D-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 and D-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 of D-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. Because D-Pro²-endomorphin-1 and D-Pro²-endomorphin-2 at high doses produced a weak antinociception with low intrinsic activity and ceiling effect, the failure for high doses of D-Pro²-endomorphin-1 or D-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, but D-Pro²-endomorphn-1 was found to be at least 2000-fold more potent than D-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 that D-Pro²-endomorphin-1 and D-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-6-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 -FNA but not by -opioid antagonist nor-BNI. On the other hand, the antinociception induced by endomorphin-2 is blocked by CTOP, -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 -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 that D-Pro²-endomorphin-1 and D-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.