Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Endomorphin-1 (Tyr-Pro-Trp-Phe-NH₂) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH₂) are endogenous opioid peptides, which selectively bind and activate µ-opioid receptors (Zadina et al., 1997). In opioid receptor binding assays, both endomorphin-1 and endomorphin-2 compete with both µ₁- and µ₂-receptor sites very potently and have no appreciable affinities for - and ₁-receptors (Goldberg et al., 1998). Endomorphin-1 and endomorphin-2 activate G protein, which is mediated by stimulation of µ-opioid receptor, because the activation is selectively blocked by the µ-opioid receptor antagonists -funaltrexamine (-FNA) and D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH₂ (CTOP), but not by the -opioid receptor antagonist naltrindole or the -opioid receptor antagonist nor-binaltrophimine (nor-BNI) in the membrane preparation obtained from mouse spinal cord or periaqueductal central gray of the rat midbrain (Narita et al., 1998, 2000). Neither endomorphin-1 nor endomorphin-2 produces any G protein activation in the pons/medulla membrane preparation obtained from the µ-opioid receptor knockout mice (Mizoguchi et al., 1999). The specific actions of endo-morphin-1 and endomorphin-2 in stimulating µ-opioid receptors found in vitro are consistent with the in vivo antinociceptive studies with the tail-flick test in mice demonstrated that endomorphin-1 or endomorphin-2 when given i.t. or i.c.v. produced potent antinociception, which was blocked by the pretreatment with µ-opioid receptor antagonists naloxone, CTOP, or -FNA (Stone et al., 1997; Goldberg et al., 1998; Tseng et al., 2000; Ohsawa et al., 2001). In µ-opioid receptor knockout mice or µ-opioid receptor-deficient CXBK mice, neither endomorphin-1 nor endomorphin-2 produces any significant antinociceptive effects (Mizoguchi et al., 1999). These findings strongly indicate that µ-opioid receptors play an essential role in mediating endomorphin-1- and endomorphin-2-induced antinociception.

Recent studies indicate that the antinociceptive effects induced by endomorphin-1 or endomorphin-2 given i.c.v. or i.t. are mediated by the stimulation of different subtypes of µ-opioid receptors. This view is supported by the findings that µ₁-opioid receptor antagonist naloxonazine or morphine-6-glucuronide antagonist 3-methoxynaltrexone blocks more effectively the antinociception induced by endomorphin-2 than by endomorphin-1 (Sakurada et al., 2000). The antinociception induced by endomorphin-1 is blocked by the pretreatment with µ-opioid receptor antagonists CTOP or -FNA, but not -opioid receptor antagonist nor-BNI. On the other hand, the antinociception induced by endomorphin-2 is blocked by CTOP, -FNA, or nor-BNI. Furthermore, the antinociception induced by endomorphin-2, but not endormorphin-1, is blocked by the pretreatment with antiserum against dynorphin A(1-17). The findings indicate that activation of the subtype of µ-opioid receptors by endomorphin-2 induces the release of dynorphin A(1-17), which subsequently stimulates the -opioid receptors for producing antinociception (Ohsawa et al., 2000, 2001; Tseng et al., 2000).

Antisense oligodeoxynucleotides (AS ODNs) have been used to knockdown gene expression and have proved to be useful pharmacological tools for studying neurotransmitter receptor activities in vitro and in vivo (Wahlestedt, 1993). The µ-opioid receptor MOR-1 was cloned after the -opioid receptor was cloned in the early 1990s (Evans et al., 1992; Chen et al., 1993). By antisense mapping of the MOR-1, 14 exons, exon-1 to -14, have been cloned and many splice variants of the MOR-1 have been identified (Rossi et al., 1997; Pan et al., 1999, 2000, 2001). Abbadie et al. (2000a) show that immunoreactivity of MOR-1, which contains exon-1, -2, -3, and -4, is observed only in the superficial laminae of spinal cord and MOR-1C, which contains exon-1, -2, -3, -7, -8, and -9, is abundant in the superficial laminae of the dorsal horn and around the central canal. The presence of MOR-1C in the superficial lamina of the spinal dorsal horn suggests that it plays a role in the pain control system. Different splice variants have been found in the central nervous system and the strikingly different distributions among different splice variants indicate that they may play different role in pain processing (Schulz et al., 1998; Abbadie et al., 2000b,c).

Morphine-induced antinociception is attenuated by pretreatment with AS ODNs directed against exon-1, -4, -6, -7, -8, -9, and -10, but is not affected by AS ODNs directed against exon-2 and -3, of MOR-1 (Neilan et al., 2001). On the other hand, the antinociception induced by morphine-6-glucuronide is attenuated by AS ODNs directed against exon-2 but is unaffected by the pretreatment with AS ODNs directed against exon-1 or exon-4 to -10 (Neilan et al., 2001). AS ODNs targeting exon-1, but not exon-2 and -4 attenuate the antinociception induced by endomorphin-1 (Sanchez-Blazquez et al., 1999). Results from the antisense mapping studies suggest that those subtypes of µ-opioid receptors originally defined as µ₁ and µ₂ in binding and pharmacological studies result from alternative slicing of MOR-1 (Pasternak and Standifer, 1995; Pasternak, 2001). Present studies were designed to determine whether i.t. pretreatment with AS ODNs directed against various exons of MOR-1 might lead to differential loss of antinociception induced by endomorphin-1 and endomorphin-2. The selective µ-opioid receptor agonist DAMGO was also included for comparison.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Animals. Male CD-1 mice weighing 25 to 30 g (Charles River Laboratories, Inc., 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. All experiments were approved by and conformed to the guidelines of the Animal Care Committee of the Medical College of Wisconsin.

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 of the heat stimulus 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 percentage of 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.t. injection of endomorphin-1, endomorphin-2, or DAMGO and T₂ was the cutoff time, which was set at 10 s. To establish the dose-response curves, at least four doses were used with 8 to 11 mice in each group.

Experimental Protocols. Intrathecal injection (i.t.) 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.

Drugs. Endomorphin-1 and endomorphin-2 were obtained from Calbiochem (La Jolla, CA). DAMGO was obtained from Bachem Biosciences (King of Prussia, PA). The endomorphin-1 and -2 for i.t. injections were dissolved in 0.9% saline containing 10% hydroxypropyl--cyclodextrin, and DAMGO was dissolved in saline containing 0.01% of Triton X-100.

Antisense Oligodeoxynucleotides. Antisense ODNs against exon-1, -4, and -8 of MOR-1 were purchased from Midland Certified Reagent Company (Midland, TX). The antisense and mismatch ODNs sequences are shown in Table 1. Mismatch sequences of exon-1 is adapted from Neilan et al. (2001). Mismatch sequences of exon-4 and -8 of MOR-1 were designed in which two and three pairs, respectively, were reversed as a measure of the sequence-specificity of the antisense oligomer. A search of GeneBank showed that the mismatch sequence was not homologous to any known nontarget genes in the mouse. The ODNs were reconstituted in sterile saline and stored at 20°C for subsequent use.

Statistical Analysis. The antinociceptive responses, %MPE, were presented as the mean ± S.E.M. A two-way analysis of variance followed by Bonferroni post tests was used to determine significance of the differences between the means. One evaluation involved determining the number of days (1-4 days) required to produce an effect by the daily pretreatment with the AS ODNs on endomorphins-induced antinociception. Another evaluation was to determine the selectivity produced by the pretreatment with AS ODNs against the antinociception produced by DAMGO. 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, San Diego, CA). The F test was used to test the difference of ED₅₀ between the endomorphins and vehicle.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Time Courses of Change of Tail-Flick Inhibition Induced by Endomorphin-1 and Endomorphin-2 Given i.t. after Once Daily i.t. Pretreatment with AS ODNs against Exon-1, -4, or -8 of MOR-1. Endomorphin-1 (16.4 nmol) and endomorphin-2 (35.0 nmol) given i.t. produced 86 to 98, %MPE of the tail-flick inhibition in mice pretreated i.t. with vehicle or the mismatch ODNs against exon-1 of MOR-1 for 24 h. Repeated daily pretreatments with vehicle or the mismatch ODNs for 2 to 4 days did not significantly affect the tail-flick inhibition induced by endomorphin-1 or endomorphin-2. However, pretreatment with AS ODNs against exon-1of MOR-1 for 24 h significantly attenuated the tail-flick inhibition induced by i.t.-administered endomorphin-1 or endomorphin-2. The attenuation of the tail-flick inhibition induced by endomorphin-1 or endomorphin-2 was time-dependent and reached its lowest level at 37, %MPE after 3 days of i.t. pretreatment with AS ODNs against exon-1 of MOR-1 (Fig. 1, A and B).

View larger version (40K): [in this window] [in a new window]   Fig. 1.   Time course of i.t. pretreatment with AS ODNs against exon-1 of MOR-1 on the inhibition of the tail-flick response induced by i.t.-administered endomorphin-1 (A) and endomorphin-2 (B), respectively. Groups of mice (8-10/group) were pretreated i.t. (5 µl each) with exon-1 AS ODNs (5 µg), mismatch ODNs (5 µg), or vehicle daily for 1, 2, 3, or 4 days before i.t. administration of endomorphin-1 (16.4 nmol) and endomorphin-2 (35.0 nmol), respectively. The peak tail-flick inhibition (%MPE) observed at 2.5, 5, or 7.5 min after i.t. administration of endomorphin-1 or endomorphin-2 was used to calculate the antinociceptive effect. Each column represents the mean and the vertical bar represents the S.E.M. Two-way ANOVA followed by Bonferroni's post test was used to test the difference between groups. *, p < 0.01; **, p < 0.001 compared with vehicle-injected groups; , p < 0.01; , p < 0.001 compared with mismatch ODN-injected groups. , vehicle; , exon-1 antisense; , exon-1 mismatch.

View larger version (45K): [in this window] [in a new window]   Fig. 2.   Time course of i.t. pretreatment with AS ODNs against exon-4 of MOR-1 on the inhibition of the tail-flick response induced by i.t. administered endomorphin-1 (A) and endomorphin-2 (B), respectively. Groups of mice (8-11/group) were pretreated i.t. (5 µl each) with exon-4 AS ODNs (5 µg), mismatch ODNs (5 µg), or vehicle daily for 1, 2, 3, or 4 days before i.t. administration of endomorphin-1 (16.4 nmol) and endomorphin-2 (35.0 nmol), respectively, and the peak tail-flick inhibition (%MPE) observed at 2.5, 5, or 7.5 min after i.t. administration of endomorphin-1 or endomorphin-2 was used to calculate the antinociceptive effect. The difference between groups was as follows: *, p < 0.001 compared with vehicle-injected group; , p < 0.01; , p < 0.001 compared with mismatch ODN-injected groups. , vehicle; , exon-4 antisense; , exon-4 mismatch.

View larger version (44K): [in this window] [in a new window]   Fig. 3.   Time course of i.t. pretreatment with AS ODNs against exon-8 of MOR-1 on the inhibition of the tail-flick response induced by i.t.-administered endomorphin-1 (A) and endomorphin-2 (B), respectively. Groups of mice (8-11/group) were pretreated i.t. (5 µl each) with exon-8 AS ODNs (5 µg), mismatch ODNs (5 µg), or vehicle daily for 1, 2, 3, or 4 days before i.t. administration of endomorphin-1 (16.4 nmol) and endomorphin-2 (35.0 nmol), respectively, and the peak tail-flick inhibition (%MPE) observed at 2.5, 5, or 7.5 min after i.t. administration of endomorphin-1 or endomorphin-2 was used to calculate the antinociceptive effect. The difference between groups was as follows: *, p < 0.05; **, p < 0.001 compared with vehicle-injected groups; , p < 0.001compared with mismatch ODN-injected groups. , vehicle; , exon-8 antisense; , exon-8 mismatch.

Dose-Response Relationships for i.t.-Administered Endomorphin-1- and Endomorphin-2-Induced Tail-Flick Inhibition in Mice Pretreated i.t. with AS ODNs against Exon-1, -4, or -8 of MOR-1 Once a Day for 3 Days. Groups of mice were pretreated i.t. with AS ODNs against exon-1, -4, or -8 of MOR-1 (5 µg/5 µl) once a day for 3 days and were injected i.t. with various doses of endomorphin-1 or endomorphin-2 24 h after the last injection of AS ODNs. Endomorphin-1 at doses from 1.6 to 16.4 nmol and endomorphin-2 at doses from 1.8 to 35.0 nmol dependently inhibited the tail-flick response in mice pretreated with vehicle. Intrathecal pretreatment with AS ODNs directed against exon-1 of MOR-1 markedly attenuated the tail-flick inhibitions induced by endomorphin-1 or endomorphin-2. The dose-response curves of endomorphin-1- and endomorphin-2-induced tail-flick inhibition were shifted to right by 4.54- and 5.33-fold, respectively, compared with the corresponding groups of mice pretreated with vehicle (Fig. 4A; Table 2).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Dose-response curves for the inhibition of the tail-flick response induced by i.t.-administered endomorphin-1 and endomorphin-2 in mice pretreated with vehicle, and AS ODNs against exon-1 (A), -4 (B), or -8 (C) of MOR-1. Groups of mice (8-10/group) were pretreated i.t. (5 µl each) with vehicle or different AS ODNs (5 µg) daily for 3 days against MOR-1 before i.t. injection with various doses of endmorphin-1 or endomorphin-2, and tail-flick responses were measured. The peak tail-flick inhibition (%MPE) observed at 2.5, 5, or 7.5 min after i.t. administration of endomorphin-1 or endomorphin-2 was used to calculate the antinociceptive effect. Each point represents the mean and the vertical bar represents the S.E.M. Nonlinear regression model was used to fit the log dose-response curve.

Tail-Flick Inhibition Induced by i.t.-Administered DAMGO in Mice Pretreated i.t. with AS ODNs against Exon-1, -4, or -8 of MOR-1 for 3 Days. Groups of mice were pretreated i.t. with AS ODNs against exon-1, -4, or -8 of MOR-1 (5 µg/5 µl) once a day for 3 days and were injected i.t. with DAMGO (0.02 nmol) 24 h after the last injection of the AS ODNs. The tail-flick responses were measured at various times after the injection. The peak tail-flick inhibition measured at 5, 10, or 15 min was used to calculate the antinociceptive effect. Other groups of mice pretreated i.t. with vehicle or mismatch ODNs against respective exon-1, -4, and -8 of MOR-1 served as controls. Intrathecal injection of 0.02 nmol of DAMGO produced 80 to 90% MPE of the tail-flick inhibition in mice pretreated with vehicle. Intrathecal pretreatment with mismatch AS ODNs against exon-1, -4, or -8 of MOR-1 did not affect the DAMGO-induced tail-flick inhibition compared with groups of mice pretreated with vehicle. However, the DAMGO-induced tail-flick inhibition was markedly attenuated by i.t. pretreatment with AS ODNs against exon-1 of MOR-1 and, to a lesser extent, by the AS ODNs against exon-8 of MOR-1 (Fig. 5).

View larger version (35K): [in this window] [in a new window]   Fig. 5.   Inhibition of the tail-flick response induced by i.t.-administered DAMGO in mice pretreated with vehicle, AS ODNs, and mismatch ODNs against exon-1, exon-4, or exon-8 of MOR-1, respectively. Groups of mice (8-10/group) were pretreated i.t. (5 µl each) with vehicle, antisense ODNs, or mismatch ODNs (5 µg) daily for 3 days against MOR-1 before i.t. injection with DAMGO, and the tail-flick responses were measured. The peak tail-flick inhibition (%MPE) observed at 5, 10, or 15 min after i.t. administration of DAMGO was used to calculate the antinociceptive effect. The difference between group: *, p < 0.01; **, p < 0.001 compared with vehicle-injected groups; , p < 0.05; , p < 0.001 compared with mismatch ODN-injected groups. , vehicle; , antisense; , mismatch.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The antinociception induced by endomorphin-1 and endomorphin-2 has been demonstrated to be selectively mediated by the stimulation of µ-opioid receptors. This is evidenced by the finding that the tail-flick inhibition induced by endomorphin-1 and endomorphin-2 when given i.t. or i.c.v. is blocked by the pretreatment with selective µ-opioid receptor antagonists naloxone, CTOP, or -FNA (Stone et al., 1997; Goldberg et al., 1998; Tseng et al., 2000; Ohsawa et al., 2001). However, recent studies indicate that the antinociceptive effects induced by endomorphin-1 and endomorphin-2 given i.c.v. or i.t. are mediated by the stimulation of different subtypes of µ-opioid receptors (Tseng et al., 2000; Ohsawa et al., 2001).

We found in the present studies that i.t. pretreatment of mice with AS ODNs against exon-1, -4, or -8 of MOR-1 differentially attenuated the antinociception induced by i.t.-administered endomorphin-1, endomorphin-2, or DAMGO. Pretreatment with AS ODNs against exon-1 of MOR-1 for 3 days was about equally effective in attenuating the antinociception induced by endomorphin-1, endomorphin-2, or DAMGO. However, pretreatment with AS ODNs against exon-4 for 3 days was more effective in attenuating the antinociception induced by endomorphin-1 and endomorphin-2, and was much less effective in attenuating the antinociception induced by DAMGO. On the other hand, pretreatment with AS ODNs against exon-8 of MOR-1 for 3 days was found to be more effective in attenuating the antinociception induced by endomorphin-1 and, to a lesser extent, by DAMGO, but showed no effect in attenuating the antinociception induced by endomorphin-2. Our results strongly suggest the presence of multiple µ-opioid receptors, which are differentially stimulated by endomorphin-1, endomorphin-2, and DAMGO to produce antinociception.

The results of our finding are consistent in part with a previous report by Sanchez-Blazquez et al. (1999), in which pretreatment with AS ODNs against exon-1, but not exon-2 or exon-4, of MOR-1 given i.c.v. attenuates the antinociception induced by endomorphin-1. We found in the present study that antinociception induced by endomorphin-1 was attenuated by i.t. pretreatment with AS ODNs against not only exon-1 but also exon-4 and -8 of MOR-1. The different effect of AS ODNs against exon-4 on endomorphin-1 between the present study and Sanchez-Blazquez et al. (1999) may be due to by different sites of administration (i.t. in our experiment versus i.c.v. in Sanchez-Blazquez's experiment), different protocol used for AS ODNs treatment, or different dose of endomorphin-1 used (16.4 nmol in the present study versus 6.5 nmol in Sanchez-Blazquez's experiment).

We found that the antinociception induced by endomorphin-2 was attenuated by i.t. pretreatment with AS ODNs against exon-1 or exon-4, but not by exon-8 of MOR-1. Rossi et al. (1996, 1997) demonstrated that antinociception induced by heroin and morphine-6-glucuronide is attenuated by AS ODNs against exon-2 or exon-3, but not by exon-1 or exon-4. The antinociception induced by morphine is attenuated by AS ODNs against exon-1 and most of the other exons except exon-2 (Neilan et al., 2001). Thus, the receptors stimulated by endomorphin-2 for producing antinociception are different from that of receptors stimulated by heroin or morphine-6-glucuronide. The results of the present study with AS ODNs against various exons of MOR-1 are in line with our previous pharmacological studies, which indicate the presence of a noval µ-opioid receptor subtype responsible for endomorphin-2 to produce antinociception (Tseng et al., 2000; Ohsawa et al., 2001; Wu et al., 2001). The multiple µ-opioid receptors for endomorphin-1, endomorphin-2, and other µ-opioids may result from alternatively spliced variants of exons of MOR-1 (Pan et al., 1999, 2001).

Antinociception induced by endomorphin-1 and endomorphin-2 seems to be mediated by the stimulation of two different subtypes of µ-opioid receptors. It has been proposed that one subtype of µ-opioid receptors is stimulated by both endomorphin-1 and endomorphin-2 and another subtype of µ-opioid receptors is stimulated solely by endomorphin-2 (Tseng et al., 2000; Ohsawa et al., 2001). This view is further supported by the findings that µ₁-opioid receptor antagonist naloxonazine blocks more effectively the antinociception induced by endomorphin-2 than endomorphin-1 and morphine-6-glucuronide antagonist 3-methoxynaltrexone blocks endomorphin-2-induced antinociception without affecting endomorphin-1-induced antinociception given i.t (Sakurada et al., 2000). There is an asymmetric cross-tolerance between endomorphin-1 and endomorphin-2 for producing antinocicpeption. Mice made acute tolerant to endomorphin-1 are not cross-tolerant to endomorphin-2, whereas mice made tolerant to endomorphin-2 are partially cross-tolerant 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).

We found in the present study that the antinociception induced by DAMGO was effectively attenuated by the pretreatment with AS ODNs against exon-1, but is much less effected by pretreatment with AS ODNs directed against exon-4 or exon-8. The results are consistent in general with the finding by others (Rossi et al., 1996; Leventhal et al., 1997; Sanchez-Blazquez et al., 1999).

It is concluded that mice pretreated i.t. with AS ODNs against exon-1, -4, or -8 of MOR-1 attenuated the antinociception induced by endomorphin-1. However, only AS ODNs against exon-1 or -4, but not exon-8, of MOR-1 attenuated the antinociception induced by endomorphin-2. The antinociception induced by DAMGO was attenuated by i.t. pretreatment of AS ODNs directed against exon-1 or -8, but not exon-4, of MOR-1. We therefore propose that distinct subtypes of µ-opioid receptors, which reflect different splice variants of exons of MOR-1, are involved in antinociceptive response induced by endomorphin-1, endomorphin-2, and DAMGO.