Introduction

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The newly isolated endogenous opioid tetrapeptides, endomorphin-1 and endomorphin-2, although consisting of only four amino acids, have high affinity and selectivity for µ-opioid receptors (Zadina et al., 1997). Both endomorphin-1 and endomorphin-2 act through µ-opioid receptors and show similar potency in opioid receptor binding and cAMP-stimulating assays (Goldberg et al., 1998; Monory et al., 2000). These endomorphins are found in the brain and spinal cord where high densities of µ-opioid receptors occur. Endomorphin-1-like immunoreactivity is more prominent in brain, whereas endomorphin-2-like immunoreactivity is more prevalent in the spinal cord (Martin-Schild et al., 1999).

In early reports, endomorphins were thought to act through both µ₁- and µ₂-receptors (Stone et al., 1997; Zadina et al., 1997; Goldberg et al., 1998; Narita et al., 1998). More recent results intimate that endomorphin-1 and endomorphin-2 might act on different subtypes of µ-opioid receptors. In isolated bronchus of guinea pig, only the effect of endomorphin-1, but not endomorphin-2, is inhibited by naloxone (Fischer and Undem, 1999). Sakurada et al. (1999, 2000) reported that pretreatment with naloxonazine blocked more effectively the antinociceptive effects induced by i.c.v. or intrathecal (i.t.) administration of endomorphin-2 than endomorphin-1. Also, 3-methoxy naltrexone blocks the antinociception induced by endomorphin-2 but not endomorphin-1, whereas -funaltrexamine inhibits both. Like other µ-receptor agonists administered supraspinally in the mouse, endomorphin-1- and endomorphin-2-induced antinociception from supraspinal sites involve spinopetal noradrenergic and serotonergic pathways. However, the supraspinal endomorphin-2- but not endomorphin-1-induced tail-flick inhibition is also blocked by i.c.v. or i.t. pretreatment with antiserum against dynorphin A(1-17) or nor-binaltorphimine or i.t. pretreatment with antiserum against Met-enkephalin or naltriben (Tseng et al., 2000). The findings indicate that supraspinal endomorphin-2-induced antinociception also contains additional components, which are mediated by the releases of dynorphin A(1-17) and Met-enkephalin acting on opioid - and ₂-receptors, respectively. Since the blockade of µ-opioid receptors by pretreatment with µ antagonists D-Phe-Cys-Tyr-Orn-Thr-Pen-Thr-NH₂ or -funaltrexamine completely block both endomorphin-1- and endomorphin-2-induced antinociception, it has been proposed that two separate subtypes of µ-opioid receptors are involved in antinociceptive effects induced by endomorphin-1 and endomorphin-2. One subtype of opioid µ-receptors is stimulated by both endomorphin-1 and endomorphin-2, and another subtype of µ-receptors is stimulated solely by endomorphin-2.

If separate µ-opioid receptors and neural mechanisms mediate the antinociception induced by endomorphin-1 and endomorphin-2, cross-tolerance might not develop between the two. Present studies were designed to determine whether i.c.v. administration of a single dose of endomorphin-1, endomorphin-2, or [D-Ala²,N-Me-Phe⁴,Gly-ol⁵]-enkephalin (DAMGO; tolerogen) could produce cross-tolerance to the subsequent antinociceptive effect of endomorphins administered i.c.v. (challenging agent).

	Materials and Methods

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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. Animals were used only once in a given experiment. All experiments were approved by and conformed to the guidelines of the Medical College of Wisconsin Animal Care Committee.

Assessment of Antinociception. Antinociceptive responses were measured with the tail-flick test (D'Amour and Smith, 1941). To measure the latency of the tail-flick response, mice were gently held with the tail put on the apparatus (Model TF6; EMDIE Instrument Co., Maidens, VA). The tail-flick response was elicited by applying radiant heat to the dorsal surface of the tail. The intensity was set to provide a predrug tail-flick response time of 3 to 4 s. The inhibition of the tail-flick response was expressed as "percent maximum possible effect (%MPE)", which was calculated as [(T₁  T₀)/(T₂  T₀)] × 100. T₀ and T₁ were the tail-flick latencies before and after i.c.v. injection of endomorphins or DAMGO, whereas T₂ was the cutoff time, which was set at 10 s. To establish the dose-response curves, at least four doses of the challenging agents were used with 7 to 11 mice at each dose. With a 50% increase of the %MPE as a quantal index of inhibition, the median antinociceptive dose (ED₅₀) and 95% confidence intervals were determined.

Experimental Protocols. Intracerebroventricular injection was performed according to the method of Haley and McCormick (1957) using a 25-µl Hamilton syringe with a 26-gauge needle. The injection volume was 4 µl. To induce acute tolerance, mice were pretreated with a single injection of endomorphin-1, endomorphin-2, DAMGO, or control vehicle. For endomorphins as tolerogens, groups of animals were pretreated with a single dose of endomorphin-1 or endomorphin-2 given i.c.v. at different times before i.c.v. challenge with a single dose of endomorphin-1 and endomorphin-2, respectively, and the tail-flick responses were measured at various times after injection. The time when the tolerance reached its maximal level after i.c.v. administration was then determined. The peak time for DAMGO pretreatment to induce acute tolerance was known to be 3 h (Narita et al., 1995).

Drugs. Endomorphin-1 (Tyr-Pro-Trp-Phe-NH₂) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH₂) were obtained from Calbiochem Corp. (La Jolla, CA). DAMGO was obtained from Bachem Biosciences Inc. (King of Prussia, PA). Endomorphins for i.c.v. injections were dissolved in 0.9% NaCl solution containing 10% hydroxypropyl--cyclodextrin; DAMGO was dissolved in 0.9% saline containing 0.01% of Triton X-100.

Statistical Analysis. The antinociceptive responses, %MPE, were presented as the mean ± S.E.M. The two-way ANOVA followed by Bonferroni post-test were used to determine the time in which the tolerance reached a maximum after endomorphin injections and tested the significance among DAMGO-pretreated groups. Nonlinear regression model was used to fit the dose-response curve. The dose-response curves, ED₅₀ values and their 95% confidence intervals, were determined by using GraphPad Prism software (version 3.0; GraphPad Software, Inc., San Diego, CA). The F test was used to test the difference of ED₅₀ between the endomorphin and vehicle.

	Results

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Time Courses of the Tail-Flick Response to i.c.v. Administration of Endomorphin-1, Endomorphin-2, and DAMGO. Groups of mice were injected i.c.v. with endomorphin-1 (30 nmol), endomorphin-2 (70 nmol), DAMGO (0.03 nmol), or vehicle, and the tail-flick responses were measured at various times after injection. The inhibition of the tail-flick response after i.c.v. administration of endomorphin-1 or endomorphin-2 developed rapidly, reached their peaks 5 min after injection, and returned to the preinjection levels 20 to 30 min after injection (Fig. 1). The inhibition of the tail-flick response after i.c.v. administration of DAMGO developed slowly, reached its peak 20 min after injection, and returned to the preinjection level 60 min after injection. Injection of saline vehicle given i.c.v. did not show any change in the latency of the tail-flick response (Fig. 1).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Time course of changes of the tail-flick response to i.c.v. administered endomorphin-1, endomorphin-2, DAMGO, and vehicle. Groups of mice were treated i.c.v. (4 µl each) with endomorphin-1 (30 nmol), endomorphin-2 (70 nmol), DAMGO (0.03 nmol), and vehicle, respectively, and the tail-flick responses were then measured after injection. The %MPE was calculated as [(T1  T0)/(T2  T0)] × 100, in which T0 and T1 were the tail-flick latencies before and after i.c.v. injection of endomorphins, DAMGO, and vehicle; T2 was the cutoff time, which was set at 10 s. Each column represents the mean, and the vertical bar represents the S.E.M. with 8 to 10 mice per group.

Time Courses of the i.c.v. Pretreatment with Endomorphin-1 or Endomorphin-2 on the Tail-Flick Inhibition Induced by Endomorphin-1 or Endomorphin-2, Respectively, Given i.c.v. Groups of mice were pretreated with endomorphin-1 (30 nmol) or endomorphin-2 (70 nmol) given i.c.v. at various times before i.c.v. injection of endomorphin-1 (15 nmol) and endomorphin-2 (40.8 nmol), respectively, and the tail-flick response was measured at various times after injection. Other groups of mice were pretreated i.c.v. with vehicle and challenged with the same dose of endomorphin-1 or endomorphin-2 to serve as controls. The i.c.v. administration of endomorphin-1 (30 nmol) or endomorphin-2 (70 nmol) produced consistent 77 to 84% MPE of the maximum tail-flick inhibition at 5 or 7.5 min after injection in mice pretreated i.c.v. with vehicle. The inhibition of the tail-flick response induced by endomorphin-1 or endomorphin-2 was attenuated time-dependently by i.c.v. pretreatment with endomorphin-1 or endomorphin-2, respectively. The attenuation of the endomorphin-1-induced tail-flick inhibition developed slowly, reached a maximal level at 2 h, and returned to control levels at 3 or 4 h. The attenuation of the endomorphin-2-induced tail-flick inhibition developed rather rapidly, reached a maximal level at 1 h, and returned to control level 90 min or 2 h after the pretreatment (Fig. 2, A and B). Pretreatment times (2 and 1 h, respectively) for endomorphin-1 and endomorphin-2 were then used for the following experiments.

View larger version (34K): [in this window] [in a new window]   Fig. 2.   Time course of i.c.v. pretreatment with endomorphin-1 (A) and endomorphin-2 (B) on the inhibition of the tail-flick response induced by i.c.v. administered endomorphin-1 and endomorphin-2, respectively. A, groups of mice were pretreated i.c.v. (4 µl each) with endomorphin-1 (30 nmol) or vehicle 1, 2, 3, or 4 h before i.c.v. administration of endomorphin-1 (15 nmol), and the tail-flick responses were then measured after injection. B, groups of mice were pretreated i.c.v. (4 µl each) with endomorphin-2 (70 nmol) or vehicle 0.5, 1, 1.5, or 2 h before i.c.v. administration of endomorphin-2, and the tail-flick responses were then measured after injection. The highest %MPE was used to calculate the tolerance effect. The %MPE was calculated as [(T1  T0)/(T2  T0)] × 100, in which T0 and T1 were the tail-flick latencies before and after i.c.v. injection of endomorphins; T2 was the cutoff time, which was set at 10 s. Each column represents the mean, and the vertical bar represents the S.E.M. with 8 to 11 mice per group. Two-way ANOVA followed by Bonferroni's post-test was used to test the difference among groups. *, p < 0.01 compared with vehicle-injected groups.

Effects of the i.c.v. Pretreatment with Endomorphin-1, Endomorphin-2, or DAMGO on the Tail-Flick Inhibition Induced by Endomorphin-1 or Endomorphin-2 Given i.c.v. Endomorphin-1 at doses between 2 and 35.8 nmol and endomorphin-2 at doses between 4 and 60 nmol given i.c.v. dose-dependently inhibited the tail-flick response in mice pretreated with vehicle for 2 and 1 h, respectively. The i.c.v. pretreatment with 30 nmol of endomorphin-1 for 2 h attenuated markedly the tail-flick inhibition induced by endomorphin-1, and the dose-response curve was shifted to the right by 3-fold compared with that of mice pretreated with vehicle (Fig. 3A, Table 1). Similarly, the i.c.v. pretreatment with 70 nmol of endomorphin-2 for 1 h attenuated markedly the tail-flick inhibition induced by endomorphin-2, and the dose-response curve was shifted to the right by 3.9-fold compared with that of mice pretreated with vehicle (Fig. 3B, Table 2). The i.c.v. pretreatment with 70 nmol of endomorphin-2 for 1 h significantly attenuated the tail-flick inhibition induced by endomorphin-1, and the dose-response curve for endomorphin-1-induced tail-flick inhibition was shifted to the right by 2.3-fold compared with that of mice pretreated with vehicle for 1 h (Fig. 3A, Table 1). However, the i.c.v. pretreatment with 30 nmol of endomorphin-1 for 2 h did not cause any significant change of the tail-flick inhibition induced by endomorphin-2; the dose-response curve and the ED₅₀ value of endomorphin-2 for the inhibition of the tail-flick response were not affected by the pretreatment with endomorphin-1 (Fig. 3B, Table 2).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Dose-response curves for the inhibition of the tail-flick response induced by i.c.v. administered endomorphin-1 (EM1; A) and endomorphin-2 (EM2; B) in mice pretreated i.c.v. with endomorphin-1, endomorphin-2, or vehicle. Groups of mice were pretreated i.c.v. (4 µl each) with endomorphin-1 at 2 h, endomorhin-2 at 1 h, or vehicle at 1 or 2 h before being injected i.c.v. with various doses of endomorphin-1 (A) or endomorphin-2 (B), and the tail-flick response was measured after injection. The highest %MPE was used to calculate the cross-tolerance effect. The %MPE was calculated as [(T1  T0)/(T2  T0)] × 100, in which T0 and T1 were the tail-flick latencies before and after i.c.v. injection of endomorphins; T2 was the cutoff time, which was set at 10 s. Each point represents the mean, and the vertical bar represents S.E.M. with 7 to 11 mice per group. Nonlinear regression model was used to fit the dose-response curve. Log scale was used as horizontal axis.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Effects of i.c.v. pretreatment with DAMGO on the tail-flick inhibition induced by endomorphin-1, endomorphin-2, or DAMGO given i.c.v. Groups of mice were pretreated i.c.v. (4 µl each) with DAMGO (0.03 nmol) or vehicle at 3 h before i.c.v. injection with endomorphin-1 (17.9 nmol), endomorphin-2 (40.8 nmol), or DAMGO (0.03 nmol), and the tail-flick responses were measured after injection. The highest %MPE was used to calculate the cross-tolerance effect. The %MPE was calculated as [(T1  T0)/(T2  T0)] × 100, in which T0 and T1 were the tail-flick latencies before and after i.c.v. injection of endomorphins or DAMGO; T2 was the cutoff time, which was set at 10 s. Each column represents the mean, and the vertical bar represents S.E.M. with 8 to 10 mice per group. Two-way ANOVA followed by Bonferroni's post-test was used to test the difference among groups. *, p < 0.05; **, p < 0.001 compared with mice pretreated with the vehicle.

	Discussion

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The antinociceptive effect induced by endomorphin-1 and endomorphin-2 has been previously demonstrated to be selectively mediated by the stimulation of µ-opioid receptors. This is evidenced by the finding that antinociception with the tail-flick test induced by endomorphin-1 and endomorphin-2 given supraspinally or spinally is blocked by the pretreatment with opioid µ-receptor antagonists -funaltrexamine or naloxone but not by - or -opioid receptor antagonists naltrindole and nor-binaltorphimine (Zadina et al., 1997; Goldberg et al., 1998; Tseng et al., 2000; Ohsawa et al., 2001).

Like other opioid µ-receptor agonists, the i.c.v. pretreatment with a high dose of endomorphin-1 and endomorphin-2 induces an acute antinociceptive tolerance to the subsequently challenging dose of i.c.v. administered endomorphin-1 and endomorphin-2, respectively. The results of our findings are consistent with the previous report by Stone et al. (1997), which demonstrated that intrathecal pretreatment with endomorphin-1 and endomorphin-2 attenuated the antinociceptive response induced by endomorphin-1 and endomorphin-2, respectively, given intrathecally. We found in the present study that the antinociceptive tolerance caused by endomorphin-1 appeared to develop much slower than that caused by endomorphin-2. This endomorphin-1-induced antinociceptive tolerance reached the maximal level at 2 h and recovered 3 to 4 h after the pretreatment with endomorphin-1, whereas endomorphin-2-induced antinociceptive tolerance developed in 1 h and recovered to control level in 90 min to 2 h. We have previously reported that endomorphin-1 given i.c.v. produced a longer duration action in tail-flick inhibition than that of endomorphin-2 (Tseng et al., 2000). The shorter duration of action of endomorphin-2-induced antinociception may be related to its rapid development of acute antinociceptive tolerance. It has been proposed that the effect and magnitude of tolerance might be affected by the efficacy of the pretreatment ligand (Paronis and Holtzman, 1994; Walker et al., 1997; Allen and Dykstra, 2000). However, both peptides at high doses produced a similar intrinsic activity in the tail-flick inhibition and increased the guanosine 5'-O -(3-[³⁵ S]thiotriphosphate) ([³⁵ S]GTP  S) bindings (Mizoguchi et al., 2000). It is unlikely that the different time courses of the development of acute antinociceptive tolerance are due to different degrees of receptor stimulation by these two peptides. Other factors such as different half-life and metabolic degradation should also be considered (Stone et al., 1997; Shane et al., 1999). Thus, different neuronal mechanisms may be involved in the induction of antinociceptive tolerance caused by endomorphin-1 and endomorphin-2.

We found in the present study that mice made tolerant to endomorphin-1 by i.c.v. pretreatment with endomorphin-1 exhibited nearly no cross-tolerance to endomorphin-2 for producing antinociception. Also, mice made tolerant to endomorphin-2 exhibited a partial cross-tolerance to endomorphin-1 for producing antinociception. Thus, the present finding adds additional evidence to support the view proposed in our previous reports that different neuronal mechanisms are involved in antinociception induced by endomorphin-1 and endomorphin-2 (Tseng et al., 2000). We propose that two different subtypes of µ-opioid receptors are involved in antinociception induced by endomorphin-1 and endomorphin-2. Thus pretreatment with endomorphin-2 still attenuates the antinociception induced by endomorphin-1; however, pretreatment with endomorphin-1 is unable to attenuate the antinociception induced by endomorphin-2. Mice made tolerant to DAMGO, a highly selective µ-opioid receptor agonist, showed cross-tolerance to endomorphin-1 but not endomorphin-2. The result indicates that endomorphin-1 and DAMGO might act on the same subtype of µ-receptor, whereas endomorphin-2 acts on another subtype of µ-receptor for producing antinociception.

We have previously demonstrated that, like morphine or DAMGO, both endomorphin-1 and endomorphin-2 given supraspinally produce their antinociception by the stimulation of µ-opioid receptors, because these antinociceptive effects induced by endomorphin-1 and endomorphin-2 given i.c.v. are blocked by the i.c.v. pretreatment with µ-opioid receptor antagonist -funaltrexamine. In addition, blockade of ₂-adrenoceptors and 5-hydroxytryptamine receptors in the spinal cord by i.t. injection of yohimbine and methysergide, respectively, effectively blocked the tail-flick inhibition induced by i.c.v. administered endomorphin-1 and endomorphin-2. However, the antinociception induced by endomorphin-2 given supraspinally contains an additional component, which is mediated by the release of dynorphin A(1-17) acting on -opioid receptors and the release of Met-enkephalin acting on ₂-opioid receptors in the spinal cord (Ohsawa et al., 2000; Tseng et al., 2000). This contention is supported by the finding that the tail-flick inhibition induced by i.c.v. administered endomorphin-2, but not endomorphin-1, is blocked by i.t. pretreatment with antiserum against dynorphin A(1-17), antiserum against Met-enkephalin, -opioid receptor antagonist nor-binaltorphimine, or ₂-opioid receptor antagonist naltriben (Ohsawa et al., 2000). The results of these studies strongly indicate that there are two separate subtypes of µ-opioid receptors involved in endomorphin-1- and endomorphin-2-induced antinociception.

It is concluded that mice made tolerant to endomorphin-2 exhibit a partial antinociceptive cross-tolerance to endomorphin-1, whereas mice made tolerant to endomorphin-1 show no cross-tolerance to endomorphin-2. We therefore propose that two separate subtypes of µ-opioid receptors are involved in antinociceptive effects induced by endomorphin-1 and endomorphin-2. One subtype of opioid µ-receptors is stimulated by DAMGO, endomorphin-1, and endomorphin-2, and another subtype of µ-receptors is stimulated solely by endomorphin-2.