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Involvement of mouse μ-opioid receptor splice variants in the spinal antinociception induced by the dermorphin tetrapeptide analog amidino-TAPA
European Journal of Pharmacology 651 (2011) 66–72
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Neuropharmacology and Analgesia
Involvement of mouse μ-opioid receptor splice variants in the spinal antinociception induced by the dermorphin tetrapeptide analog amidino-TAPA Hirokazu Mizoguchi a, Chizuko Watanabe a, Takayuki Higashiya a, Satoshi Takeda a, Kaori Moriyama a, Akihiko Yonezawa a, Takumi Sato b, Takaaki Komatsu c, Tsukasa Sakurada c, Shinobu Sakurada a,⁎ a b c
Department of Physiology and Anatomy, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan Department of Pharmacology, Nihon Pharmaceutical University, 10281 Komuro, Ina-machi, Kitaadachi-gun, Saitama 362-0806, Japan First Department of Pharmacology, Daiichi College of Pharmaceutical Sciences, 22-1 Tamagawa-cho, Minami-ku, Fukuoka 815-8511, Japan
a r t i c l e
i n f o
Article history: Received 20 February 2010 Received in revised form 2 October 2010 Accepted 16 October 2010 Available online 31 October 2010 Keywords: Antinociception Acute tolerance mu-Opioid receptor Opioid peptides Splice variants Spinal cord
a b s t r a c t The involvement of the mouse μ-opioid receptor (mMOR-1) splice variants in the antinociceptive effect of intrathecally (i.t.) administered Nα-amidino-Tyr-D-Arg-Phe-β-Ala (amidino-TAPA) and [D-Ala2,N-MePhe4, Gly-ol5]enkephalin (DAMGO) was investigated in mice by monitoring the recovery from acute antinociceptive tolerance to amidino-TAPA and DAMGO. A single i.t. pretreatment with DAMGO produced an acute antinociceptive tolerance, which peaked at 2 h and disappeared within 5 h after the pretreatment. In contrast, a single i.t. pretreatment with amidino-TAPA produced an acute antinociceptive tolerance, which disappeared within 3 h after the pretreatment. The concomitant i.t. pretreatment with an antisense oligodeoxynucleotide (ODN) for exon-1, exon-12, exon-13 or exon-14 of mMOR-1 maintained the acute antinociceptive tolerance to amidino-TAPA for 24 h after the pretreatment. On the other hand, the concomitant i.t. pretreatment with an antisense ODN for exon-1 of mMOR-1, but not an antisense ODN for exon-12, exon-13 or exon-14 of mMOR-1, maintained the acute antinociceptive tolerance to DAMGO for 24 h after the pretreatment. The present results suggest that the spinal antinociception of amidino-TAPA is partially mediated through the activation of the amidino-TAPA-sensitive and DAMGO-insensitive mMOR-1 splice variants MOR-1J, MOR-1K and MOR-1L, which contain the sequence encoded by exon-12, exon-13 and exon-14, respectively. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Nα-Amidino-Tyr-D-Arg-Phe-β-Ala (amidino-TAPA) is an N-terminal tetrapeptide derivative of dermorphin. It has a very high affinity and selectivity for μ-opioid receptors, and produces an extremely potent and long-lasting antinociception (Mizoguchi et al., 2007; Ogawa et al., 2002). The antinociceptive potency of amidino-TAPA is 10-fold, 1.2-fold or 650-fold higher than that of morphine after subcutaneous, oral or intrathecal (i.t.) injections, respectively. Unlike the traditional μ-opioid receptor agonist DAMGO, the antinociceptive effect of i.t.-administered amidino-TAPA is mediated through the release of the endogenous κ-opioid peptides dynorphin A, dynorphin B and α-neo-endorphin and the endogenous δ-opioid peptide [Leu5] enkephalin via activation of μ-opioid receptors in the spinal cord (Mizoguchi et al., 2007). The distinct antinociceptive profiles of amidino-TAPA may be mediated by the activation of distinct μ-opioid receptors that are sensitive to amidino-TAPA but insensitive to DAMGO.
⁎ Corresponding author. Tel.: +81 22 727 0124; fax: +81 22 727 0125. E-mail address: [email protected] (S. Sakurada). 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.10.049
Since the initial cloning of the μ-opioid receptor gene MOR-1 (Chen et al., 1993; Wang et al., 1993), 33 splice variants have been identified in mouse MOR-1 (mMOR-1) mRNA (Doyle et al., 2007a,b; Kvam et al., 2004; Pan et al., 2001, 2005). The sequence variability of the splice variants is found in the sequences of the N-terminal and first transmembrane region, and in the sequence of the intracellular Cterminal region. The selectivities and intrinsic activities of various μopioid receptor agonists for 6 of the splice variants (MOR-1, 1A, 1C, 1D, 1E, and 1F), that contain the sequences encoded by exon-1, have been described in CHO cells over-expressing each of these splice variants (Pasternak, 2004). However, no remarkable differences in the selectivity and intrinsic activity of any μ-opioid receptor agonist were observed among the splice variants examined. For the other splice variants, the selectivity and intrinsic activity of μ-opioid receptor agonists is still unknown. Although the distribution of most of the splice variants in the rodent central nervous system have been described (Doyle et al., 2007a,b; Pan et al., 1999, 2000, 2001, 2005), the physiological roles of each splice variant are still unknown. A single intracerebroventricular or i.t. injection of opioid receptor agonists induces a rapid and short-term reduction of their antinociceptive effect, the so-called “acute antinociceptive tolerance” (Narita et al., 1995, 1996; Suh and Tseng, 1990; Wu et al., 2001, 2003). The development of acute antinociceptive tolerance is considered to
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be a receptor-dependent phenomenon. We have previously reported that the acute antinociceptive tolerance to a δ-opioid receptor agonist is caused by the internalization and degradation of the δ-opioid receptor following the single treatment with a δ-opioid receptor agonist (Narita et al., 1996, 1997a,b). The recovery from the acute antinociceptive tolerance to a δ-opioid receptor agonist is produced by newly synthesized δ-opioid receptors, which are associated with the natural turnover of δ-opioid receptors. Therefore, the suppression of the synthesis of new δ-opioid receptors can maintain the acute antinociceptive tolerance to an δ-opioid receptor agonist. The present study was conducted to identify the mMOR-1 splice variants involved in the spinal antinociceptive effect of amidino-TAPA, but not DAMGO, by monitoring the recovery from acute antinociceptive tolerance to amidino-TAPA and DAMGO.
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following sequences: 5’-GCC CAC TAC ACA CAC GAT AGA-3’ (antisense ODN for exon-1), 5’-GTG AAG CAC TGT CTT CTA AAG GGG-3’ (antisense ODN for exon-12), 5’-TCA GTC TTT ATC AGC TCA CCG CCA-3’ (antisense ODN for exon-13) and 5’-TAT TTT GAT TTT CAG TTG ATT C-3’ (antisense ODN for exon-14). 2.5. Statistical analyses The statistical significance of differences between the groups was assessed with a one-way analysis of variance (ANOVA) and a two-way ANOVA followed by Dunnett's test and Bonferroni's test, respectively. 3. Results 3.1. Acute antinociceptive tolerance to DAMGO and amidino-TAPA
2. Materials and methods All experiments were performed following the approval of the Ethics Committee for Animal Experiments at Tohoku Pharmaceutical University and according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Every effort was made to minimize the number and any suffering of the animal used in the following experiments. 2.1. Animals Male ddY mice (Japan SLC, Hamamatsu, Japan) weighing 22–25 g were used. The animals were housed in a room maintained at 22– 23 °C and 50–60% relative humidity with an alternating 12-h light/ dark cycle. Food and water were available ad libitum. Mice were used only once. 2.2. Assessment of antinociception Antinociception was determined with the tail-flick test (D'Amour and Smith, 1941). For measurement of the latency of the tail-flick response, mice were gently held by hand with their tail positioned in an apparatus (Ugo Basile, Italy) for radiant heat stimulation on the ventral surface of the tail. The intensity of the heat stimulus was adjusted so that the animal withdrew its tail after 2.5–3.5 s. The inhibition of the tail-flick response was expressed as the percent maximal possible effect, % MPE, which was calculated as follows: [(T1 − T0)/(T2 − T0)]× 100, where T0 and T1 are the tail-flick latencies before and after the treatments, respectively, and T2 is the cutoff time, set at 10 s to avoid injury to the tail. The antinociceptive effect was represented as the mean ± S.E.M. for at least 10 mice. The dose–response curves and ED50 values with their 95% confidence intervals were calculated with a computer-assisted curve-fitting program (GraphPad Prism; GraphPad Software, Inc., San Diego, CA).
Groups of mice were injected i.t. with DAMGO (10 pmol), amidino-TAPA (2 pmol) or aCSF, and the antinociceptive effects induced by DAMGO and amidino-TAPA were measured. The i.t. administration of DAMGO or amidino-TAPA resulted in a marked antinociception with approximately equal efficacy (87.54% and 85.38% MPE for DAMGO and amidino-TAPA, respectively, at their peak-effect time) at the doses used (Fig. 1). The antinociceptive effect of amidino-TAPA developed rapidly, reached its maximal effect at 10 min after the treatment, and then disappeared slowly by 120 min. A more prolonged antinociceptive effect was observed for amidinoTAPA than for DAMGO, whose antinociceptive effect peaked at 10 min and disappeared by 45 min after the treatment. In the following experiments, to identify the acute antinociceptive tolerance to DAMGO or amidino-TAPA, the antinociceptive effects of i.t.-administered DAMGO (10 pmol) or amidino-TAPA (2 pmol) were measured 10 min after the treatment in mice receiving the various pretreatments. Groups of mice were pretreated i.t. with aCSF or DAMGO (10 pmol) 1, 2, 3, 4, 5, or 6 h before the i.t. treatment with DAMGO (10 pmol), and the antinociception induced by DAMGO was measured 10 min after the treatment. The mice pretreated with DAMGO 1 h before showed an antinociceptive effect of DAMGO treatment that was equipotent to mice pretreated with aCSF (Fig. 2A). However, in mice pretreated with DAMGO 2 h before, the antinociceptive effect of DAMGO was dramatically suppressed compared with mice pretreated
2.3. Intrathecal injection The i.t. injection was performed following the method described by Hylden and Wilcox (1980) using a 29-gauge stainless-steel needle attached to a 50-μl Hamilton microsyringe. For i.t. injections, drugs were dissolved in sterile artificial cerebrospinal fluid (aCSF) containing 126.6 mM NaCl, 2.5 mM KCl, 2.0 mM MgCl2, and 1.3 mM CaCl2. The volume for the i.t. injections was 5 μl. 2.4. Drugs The drugs used were amidino-TAPA (Peptides Institute, Osaka, Japan) and DAMGO (Sigma-Aldrich, St. Louis, MO). Antisense phosphorothioate-linked oligodeoxynucleotides (ODNs) for exon-1, exon-12, exon-13, and exon-14 of mMOR-1, which were purchased from The Midland Certified Reagent Company (Midland, TX), had the
Fig. 1. Antinociceptive effects of DAMGO and amidino-TAPA in the ddY mouse spinal cord. Groups of mice were injected i.t. with DAMGO (10 pmol) or amidino-TAPA (2 pmol), and the tail-flick inhibition induced by DAMGO or amidino-TAPA was measured for 90 or 180 min, respectively. The antinociceptive effect was calculated as the mean ± S.E.M for at least 10 mice. The statistical significance of the differences between the groups was assessed with a two-way ANOVA followed by Bonferroni's test. The F values of the two-way ANOVA for DAMGO and amidino-TAPA against aCSF were F[1,126] = 134.7 (P b 0.001) and F[1,180] = 340.4 (P b 0.001), respectively. ***P b 0.001 vs. the aCSF-treated group.
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antinociceptive tolerance to amidino-TAPA 3 h after the pretreatment was determined under conditions where the new synthesis of most of the mMOR-1 splice variants had been suppressed by the pretreatment with an antisense ODN for exon-1 of mMOR-1. Groups of mice pretreated i.t. with aCSF or an antisense ODN for exon-1 of mMOR-1 (5 μg) 24 h before, were injected i.t. with aCSF or amidino-TAPA (2 pmol) 3 h before the i.t. treatment with amidino-TAPA (2 pmol), and the tail-flick inhibition induced by amidino-TAPA was measured at 10 min after the treatments. Like the data presented in Fig. 2B, no suppression of amidino-TAPA-induced antinociception at 3 h after the pretreatment with amidino-TAPA (acute antinociceptive tolerance to amidino-TAPA) was observed in mice pretreated with aCSF 24 h before (data not shown). However, in mice pretreated with an antisense ODN for exon-1 of mMOR-1 24 h before, a marked suppression of amidino-TAPA-induced antinociception at 3 h after the pretreatment with amidino-TAPA (the acute antinociceptive tolerance to amidino-TAPA) was observed.
3.2. Retention of the acute antinociceptive tolerance to DAMGO or amidino-TAPA by co-pretreatment with exon-specific antisense ODNs for mMOR-1
Fig. 2. Acute antinociceptive tolerance to DAMGO and amidino-TAPA in the ddY mouse spinal cord. (A) Groups of mice were pretreated i.t. with aCSF or DAMGO (10 pmol) 1, 2, 3, 4, 5, or 6 h before the i.t. treatment with DAMGO (10 pmol), and the antinociception induced by DAMGO was measured 10 min after the treatment. (B) Groups of mice were pretreated i.t. with aCSF or amidino-TAPA (2 pmol) 3, 4, 5, 6, 7, or 9 h before the i.t. treatment with amidino-TAPA (2 pmol), and the antinociception induced by amidinoTAPA was measured 10 min after the treatment. The antinociceptive effect was calculated as the mean ± S.E.M for at least 10 mice. The statistical significance of the differences between the groups was assessed with a two-way ANOVA followed by Bonferroni's test. (A) The F value of the two-way ANOVA was F[1,124] = 21.90 (P b 0.001). *P b 0.05, ***P b 0.001 vs. the aCSF-pretreated group. (B) The F value of the two-way ANOVA was F[1,117] = 0.3782 (P = 0.5397).
with aCSF. The suppression of DAMGO-induced antinociception (acute antinociceptive tolerance to DAMGO) gradually disappeared. At 5 h after the pretreatment with DAMGO, the antinociceptive effect of DAMGO treatment had completely recovered to the same magnitude observed in mice pretreated with aCSF. Other groups of mice were pretreated i.t. with aCSF or amidinoTAPA (2 pmol) 3, 4, 5, 6, 7, or 9 h before the i.t. treatment with amidino-TAPA (2 pmol), and the antinociception induced by amidinoTAPA was measured 10 min after the treatment. In contrast to DAMGO, the antinociceptive effect of amidino-TAPA in mice pretreated with amidino-TAPA was exactly equipotent with that in mice pretreated with aCSF at all time points measured after the pretreatment (Fig. 2B). In other words, no suppression of amidinoTAPA-induced antinociception after the pretreatment with amidinoTAPA (acute antinociceptive tolerance to amidino-TAPA) was observed at any time point tested. Together with the evidence that the antinociceptive effect of amidino-TAPA is long-lasting and it takes 2 h to return the nociceptive threshold to the baseline level after the treatment, the present results suggests the possibility that the acute antinociceptive tolerance to amidino-TAPA develops rapidly and disappears within 3 h. To investigate this possibility, the acute
The mMOR-1 splice variants involved in the acute antinociceptive tolerance to DAMGO or amidino-TAPA in the mouse spinal cord were determined using the exon-specific antisense ODNs for mMOR-1. Groups of mice were pretreated i.t. with aCSF, an antisense ODN for exon-1 of mMOR-1 (5 μg), μ-opioid receptor agonists (10 pmol of DAMGO or 2 pmol of amidino-TAPA), or μ-opioid receptor agonists in combination with an antisense ODN for exon-1 of mMOR-1 24 h before the i.t. treatment with μ-opioid receptor agonists (10 pmol of DAMGO or 2 pmol of amidino-TAPA), and the tail-flick inhibition induced by μ-opioid receptor agonists was measured at 10 min after the treatments. The pretreatment with an antisense ODN for exon-1 of mMOR-1 24 h before did not alter the antinociception induced by either DAMGO or amidino-TAPA (Fig. 3). The antinociception induced by DAMGO and amidino-TAPA also was not affected by the 24h pretreatment with DAMGO and amidino-TAPA, respectively. The acute antinociceptive tolerance to DAMGO and amidino-TAPA was not observed 24 h after the pretreatment with DAMGO and amidinoTAPA, respectively. In contrast, the antinociception induced by DAMGO or amidino-TAPA was significantly attenuated by the concomitant pretreatment with the antisense ODN for exon-1 of mMOR-1 and either DAMGO or amidino-TAPA, respectively. The acute antinociceptive tolerance to DAMGO or amidino-TAPA was still maintained for 24 h after the pretreatment by the concomitant pretreatment with the antisense ODN for exon-1 of mMOR-1. The involvement of MOR-1J (splice variant containing the sequence encoded by exon-12), MOR-1K (splice variant containing the sequence encoded by exon-13) and MOR-1L (splice variant containing the sequence encoded by exon-14) in the acute antinociceptive tolerance to DAMGO or amidino-TAPA in the mouse spinal cord was also investigated on the same experimental schedule, using antisense ODNs for exon-12, exon-13 and exon-14 of mMOR-1, respectively. The pretreatment with antisense ODNs for either exon-12, exon-13 or exon-14 of mMOR-1 24 h before did not alter the antinociception induced by either DAMGO or amidino-TAPA (Figs. 4–6). AmidinoTAPA-induced antinociception was significantly attenuated by the concomitant pretreatment with amidino-TAPA and antisense ODNs for either exon-12, exon-13 or exon-14 of mMOR-1 (Figs. 4B, 5B, 6B), whereas DAMGO-induced antinociception was not altered by the concomitant pretreatment with DAMGO and antisense ODNs for either exon-12, exon-13 or exon-14 of mMOR-1 (Figs. 4A, 5A, 6A). The acute antinociceptive tolerance to amidino-TAPA, but not to DAMGO, was maintained for 24 h after the pretreatment by antisense ODNs for either exon-12, exon-13 or exon-14 of mMOR-1.
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Fig. 3. Effects of an antisense ODN for exon-1 of mMOR-1 on the acute antinociceptive tolerance to DAMGO and amidino-TAPA in the ddY mouse spinal cord. (A) Groups of mice were pretreated i.t. with aCSF, an antisense ODN for exon-1 of mMOR-1 (AS/Exon1: 5 μg), DAMGO (10 pmol) or DAMGO in combination with AS/Exon-1 24 h before the i.t. treatment with DAMGO (10 pmol), and the tail-flick inhibition induced by DAMGO was measured at 10 min after the treatments. (B) Groups of mice were pretreated i.t. with aCSF, AS/Exon-1 (5 μg), amidino-TAPA (2 pmol) or amidino-TAPA in combination with AS/Exon-1 24 h before the i.t. treatment with amidino-TAPA (2 pmol), and the tail-flick inhibition induced by amidino-TAPA was measured at 10 min after the treatments. The antinociceptive effect was calculated as the mean ± S.E.M for at least 10 mice. The statistical significance of the differences between the groups was assessed with a one-way ANOVA followed by Dunnett's test. (A) The F value of the one-way ANOVA was F[3,35] = 8.836 (P b 0.001). **P b 0.01 vs. the aCSF-pretreated group. (B) The F value of the one-way ANOVA was F[3,36] = 4.271 (P b 0.05). *P b 0.05 vs. the aCSFpretreated group.
4. Discussion Amidino-TAPA has a very high affinity and selectivity for μ-opioid receptors, and shows an extremely potent and long-lasting antinociception that is mediated by μ-opioid receptors (Mizoguchi et al., 2007; Ogawa et al., 2002). Unlike the traditional μ-opioid receptor agonist DAMGO, amidino-TAPA has a distinct spinal antinociceptive mechanism that is mediated by the release of the endogenous δ-opioid peptide [Leu5]enkephalin and the endogenous κ-opioid peptides dynorphin A, dynorphin B and α-neo-endorphin, which is initiated by the activation of spinal μ-opioid receptors (Mizoguchi et al., 2007). An antinociception that is mediated through the release of endogenous opioid peptides in the spinal cord is also observed for the other selective μ-opioid receptor agonists endomorphin-2 (Mizoguchi et al., 2006b; Sakurada et al., 2001), Tyr-D-Arg-Phe-Sar (TAPS: Mizoguchi et al., 2006a) and dimethyl-Tyr-D-Arg-Phe-Lys-NH2
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Fig. 4. Effects of an antisense ODN for exon-12 of mMOR-1 on the acute antinociceptive tolerance to DAMGO and amidino-TAPA in the ddY mouse spinal cord. (A) Groups of mice were pretreated i.t. with aCSF, an antisense ODN for exon-12 of mMOR-1 (AS/ Exon-12: 5 μg), DAMGO (10 pmol) or DAMGO in combination with AS/Exon-12 24 h before the i.t. treatment with DAMGO (10 pmol), and the tail-flick inhibition induced by DAMGO was measured at 10 min after the treatments. (B) Groups of mice were pretreated i.t. with aCSF, AS/Exon-12 (5 μg), amidino-TAPA (2 pmol) or amidino-TAPA in combination with AS/Exon-12 24 h before the i.t. treatment with amidino-TAPA (2 pmol), and the tail-flick inhibition induced by amidino-TAPA was measured at 10 min after the treatments. The antinociceptive effect was calculated as the mean± S.E.M for at least 10 mice. The statistical significance of the differences between the groups was assessed with a one-way ANOVA followed by Dunnett's test. (A) The F value of the one-way ANOVA was F[3,36]= 0.1393 (P= 0.9358). (B) The F value of the one-way ANOVA was F [3,76]= 3.444 (P b 0.05). *P b 0.05 vs. the aCSF-pretreated group.
([Dmt1]DALDA: Szeto et al., 2003). The evidence clearly suggests that these selective μ-opioid receptor agonists stimulate different μ-opioid receptors than DAMGO to produce the antinociception. At present, 33 splice variants for mMOR-1 have been identified (Doyle et al., 2007a, b; Kvam et al., 2004; Pan et al., 2001, 2005). The release of endogenous opioid peptides by the above μ-opioid receptor agonists may be mediated by the activation of specific splice variants of mMOR-1 that are insensitive to DAMGO. In the present study, the mMOR-1 splice variants involved in the spinal antinociception of amidino-TAPA, but not DAMGO, were identified by monitoring the recovery from the acute antinociceptive tolerance to amidino-TAPA. Acute antinociceptive tolerance is the rapid and temporary reduction of the antinociceptive effect of opioid receptor agonists observed after a single pretreatment with the same agonist. In the present study, the pretreatment with DAMGO caused the reduction of its antinociceptive effect (acute antinociceptive tolerance), which peaked at 2 h and disappeared by 5 h after the pretreatment. The
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Fig. 5. Effects of an antisense ODN for exon-13 of mMOR-1 on the acute antinociceptive tolerance to DAMGO and amidino-TAPA in the ddY mouse spinal cord. (A) Groups of mice were pretreated i.t. with aCSF, an antisense ODN for exon-13 of mMOR-1 (AS/ Exon-13: 5 μg), DAMGO (10 pmol) or DAMGO in combination with AS/Exon-13 24 h before the i.t. treatment with DAMGO (10 pmol), and the tail-flick inhibition induced by DAMGO was measured at 10 min after the treatments. (B) Groups of mice were pretreated i.t. with aCSF, AS/Exon-13 (5 μg), amidino-TAPA (2 pmol) or amidino-TAPA in combination with AS/Exon-13 24 h before the i.t. treatment with amidino-TAPA (2 pmol), and the tail-flick inhibition induced by amidino-TAPA was measured at 10 min after the treatments. The antinociceptive effect was calculated as the mean± S.E.M for at least 10 mice. The statistical significance of the differences between the groups was assessed with a one-way ANOVA followed by Dunnett's test. (A) The F value of the one-way ANOVA was F[3,36]=0.4406 (P= 0.7253). (B) The F value of the one-way ANOVA was F [3,36]=5.065 (Pb 0.01). **P b 0.01 vs. the aCSF-pretreated group.
duration of acute antinociceptive tolerance to opioid receptor agonists is variable depending on the subtype of the opioid receptors. Unlike the μ-opioid receptor agonist DAMGO, the acute antinociceptive tolerance to the δ-opioid receptor agonist [D-Ala2]deltorphin II and the κ-opioid receptor agonist U-50,488H both peaked at 3–6 h, but disappeared by 12 h and 24 h after the pretreatment, respectively (Narita et al., 1997a; unpublished observation). The development of acute antinociceptive tolerance to δ-opioid receptor agonist is caused by the internalization and degradation of δ-opioid receptors, and the recovery from the acute antinociceptive tolerance is produced by the newly synthesized δ-opioid receptors (Narita et al., 1997a). The duration of the acute antinociceptive tolerance to opioid receptor agonists may depend on the turnover speed of the opioid receptor, which is identical for each opioid receptor subtype. In the present study, the pretreatment with amidino-TAPA did not show any reduction of its antinociceptive effect (acute antinociceptive tolerance) at 3–9 h after the pretreatment. The antinociceptive effect of amidino-TAPA is long-lasting and it takes 2 h to completely return the
Fig. 6. Effects of an antisense ODN for exon-14 of mMOR-1 on the acute antinociceptive tolerance to DAMGO and amidino-TAPA in the ddY mouse spinal cord. (A) Groups of mice were pretreated i.t. with aCSF, an antisense ODN for exon-14 of mMOR-1 (AS/ Exon-14: 5 μg), DAMGO (10 pmol) or DAMGO in combination with AS/Exon-14 24 h before the i.t. treatment with DAMGO (10 pmol), and the tail-flick inhibition induced by DAMGO was measured at 10 min after the treatments. (B) Groups of mice were pretreated i.t. with aCSF, AS/Exon-14 (5 μg), amidino-TAPA (2 pmol) or amidino-TAPA in combination with AS/Exon-14 24 h before the i.t. treatment with amidino-TAPA (2 pmol), and the tail-flick inhibition induced by amidino-TAPA was measured at 10 min after the treatments. The antinociceptive effect was calculated as the mean± S.E.M for at least 10 mice. The statistical significance of the differences between the groups was assessed with a one-way ANOVA followed by Dunnett's test. (A) The F value of the one-way ANOVA was F[3,36]= 0.1991 (P= 0.8963). (B) The F value of the one-way ANOVA was F [3,36]= 6.497 (P b 0.01). **P b 0.01 vs. the aCSF-pretreated group.
nociceptive threshold to the baseline level after the treatment. Based on the above evidence, the present results suggest the possibility that the acute antinociceptive tolerance to amidino-TAPA develops rapidly and disappears within 3 h. To investigate this possibility, the acute antinociceptive tolerance to amidino-TAPA at 3 h after the pretreatment was determined under conditions where the new synthesis of most of the mMOR-1 splice variants had been suppressed by the pretreatment with antisense ODN for exon-1 of mMOR-1. Under these conditions, amidino-TAPA showed a marked acute antinociceptive tolerance, suggesting that the acute antinociceptive tolerance to amidino-TAPA developed rapidly and disappeared within 3 h. The suggestion that the duration of acute antinociceptive tolerance to amidino-TAPA is much shorter than that to DAMGO is another piece of evidence to support the hypothesis that amidino-TAPA stimulates different μ-opioid receptors than DAMGO.
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By monitoring the recovery from the acute antinociceptive tolerance to DAMGO and amidino-TAPA, the involvement of mMOR1 splice variants in the acute antinociceptive tolerance to DAMGO or amidino-TAPA was determined using exon-specific antisense ODNs for mMOR-1. In general, the prolonged pretreatment (usually 4 days) with antisense ODNs for opioid receptors is required to desensitize the opioid receptors (Narita et al., 1997b). Because antisense ODNs for the opioid receptor itself do not have any effect on existing opioid receptors on the cell membrane surface, it should take 3–4 days to desensitize existing opioid receptors with endogenous opioid peptides. In fact, a single 24-h pretreatment with an antisense ODN for exon-1 of mMOR-1 did not alter DAMGO-induced antinociception, although a 4-day pretreatment with the same antisense ODN significantly suppressed the DAMGO-induced antinociception (unpublished observation). However, a single 24-h but concomitant pretreatment with an antisense ODN for exon-1 of mMOR-1 and DAMGO suppressed DAMGO-induced antinociception, suggesting that the suppression of the synthesis of new mMOR-1 splice variants containing the sequence encoded by exon-1 can maintain the acute antinociceptive tolerance to DAMGO for 24 h after the pretreatment, which usually disappeared within 5 h after the pretreatment. This is a simple but accurate method for desensitizing a specific subtype of opioid receptor with a single pretreatment. In the present study, the concomitant pretreatment with an antisense ODN for exon-1 of mMOR-1 and amidino-TAPA maintained the acute antinociceptive tolerance to amidino-TAPA for 24 h after the pretreatment, suggesting that the splice variants of mMOR-1 that are desensitized by pretreatment with amidino-TAPA, as well as DAMGO, are the splice variants containing the sequence encoded by exon-1. Most of the splice variants for mMOR-1 identified contain the sequence encoded by exon-1 (Doyle et al., 2007a,b; Kvam et al., 2004; Pan et al., 2001, 2005). Pasternak and his colleagues described the selectivity and intrinsic activity of various μ-opioid receptor agonists for mMOR-1 splice variants containing the sequences encoded by exon-1 (Bolan et al., 2004; Pan et al., 1999, 2000; Pasternak, 2004). However, no remarkable differences in the selectivity and intrinsic activity of any μopioid receptor agonists were observed among the mMOR-1 splice variants examined. Therefore, in the present study, we focused on the other mMOR-1 splice variants, MOR-1J, MOR-1K or MOR-1L, which contain the sequence encoded by exon-12, exon-13 or exon-14, respectively. We found that amidino-TAPA-induced antinociception is significantly suppressed by a 24-h concomitant pretreatment with amidino-TAPA and an antisense ODN for either exon-12, exon-13 or exon-14 of mMOR-1. The suppression of the new synthesis of MOR-1J, MOR-1K or MOR-1L can maintain the acute antinociceptive tolerance to amidino-TAPA for 24 h after the pretreatment, suggesting that the splice variants of mMOR-1 desensitized by pretreatment with amidino-TAPA are MOR-1J, MOR-1K and MOR-1L. In contrast, DAMGO-induced antinociception was not affected by a 24-h concomitant pretreatment with DAMGO and an antisense ODN for either exon-12, exon-13 or exon-14 of mMOR-1, suggesting that MOR-1J, MOR-1K and MOR-1L are not the splice variants desensitized by pretreatment with DAMGO. In other words, these splice variants are amidino-TAPA-sensitive and DAMGO-insensitive mMOR-1 splice variants. At present, the physiological roles of the 33 identified splice variants of mMOR-1 are still unclear. We have previously found that, unlike DAMGO, amidino-TAPA induces the release of the endogenous κ-opioid peptides dynorphin A, dynorphin B and α-neo-endorphin and the endogenous δ-opioid peptide [Leu5]enkephalin in the spinal cord to produce the antinociception. In the present study, we have identified the amidino-TAPA-sensitive and DAMGO-insensitive mMOR-1 splice variants, MOR-1J, MOR-1K and MOR-1L, in the spinal cord. Together with the previous observations, the present results suggest the possibility that the release of endogenous opioid peptides by amidino-TAPA is mediated by the amidino-TAPA-sensitive and
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DAMGO-insensitive mMOR-1 splice variants MOR-1J, MOR-1K and MOR-1L.
5. Conclusion In conclusion, the spinal antinociception of amidino-TAPA is partially mediated through the activation of the distinct mMOR-1 splice variants MOR-1J, MOR-1K or MOR-1L, which contain the sequence encoded by exon-12, exon-13 or exon-14, respectively. These splice variants are amidino-TAPA-sensitive and DAMGOinsensitive mMOR-1 splice variants, which may be responsible for the distinct antinociceptive properties of amidino-TAPA.
Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research (C) [KAKENHI 21600013 and 22600009] from the Japan Society for the Promotion of Science, and a Matching Fund Subsidy for Private Universities from the Ministry of Education, Culture, Sports, Science, and Technology Japan (2010–2014).
References Bolan, E.A., Pan, Y.X., Pasternak, G.W., 2004. Functional analysis of MOR-1 splice variants of the mouse mu opioid receptor gene Oprm. Synapse 51, 11–18. Chen, Y., Mestek, A., Liu, J., Hurley, A., Yu, L., 1993. Molecular cloning and functional expression of a μ-opioid receptor from rat brain. Mol. Pharmacol. 44, 8–12. D'Amour, F.E., Smith, D.L., 1941. A method for determining loss of pain sensation. J. Pharmacol. Exp. Ther. 72, 74–79. Doyle, G.A., Sheng, X.R., Lin, S.S., Press, D.M., Grice, D.E., Buono, R.J., Ferraro, T.N., Berrettini, W.H., 2007a. Identification of three mouse μ-opioid receptor (MOR) gene (Oprm1) splice variants containing a newly identified alternatively spliced exon. Gene 388, 135–147. Doyle, G.A., Sheng, X.R., Lin, S.S., Press, D.M., Grice, D.E., Buono, R.J., Ferraro, T.N., Berrettini, W.H., 2007b. Identification of five mouse μ-opioid receptor (MOR) gene (Oprm1) splice variants containing a newly identified alternatively spliced exon. Gene 395, 98–107. Hylden, J.L., Wilcox, G.L., 1980. Intrathecal morphine in mice: a new technique. Eur. J. Pharmacol. 67, 313–316. Kvam, T.M., Baar, C., Rakvåg, T.T., Kaasa, S., Krokan, H.E., Skorpen, F., 2004. Genetic analysis of the murine μ opioid receptor: increased complexity of Oprm gene splicing. J. Mol. Med. 82, 250–255. Mizoguchi, H., Ito, K., Watanabe, H., Watanabe, C., Katsuyama, S., Fujimura, T., Sakurada, T., Sakurada, S., 2006a. Contribution of spinal μ1-opioid receptors and dynorphin B to the antinociception induced by Tyr-D-Arg-Phe-Sar. Peptides 27, 2786–2793. Mizoguchi, H., Watanabe, H., Hayashi, T., Sakurada, W., Sawai, T., Fujimura, T., Sakurada, T., Sakurada, S., 2006b. Possible involvement of dynorphin A (1–17) release via μ1opioid receptors in spinal antinociception by endomorphin-2. J. Pharmacol. Exp. Ther. 317, 362–368. Mizoguchi, H., Watanabe, C., Watanabe, H., Moriyama, K., Sato, B., Ohwada, K., Yonezawa, A., Sakurada, T., Sakurada, S., 2007. Involvement of endogenous opioid peptides in the antinociception induced by the novel dermorphin tetrapeptide analog amidino-TAPA. Eur. J. Pharmacol. 560, 150–159. Narita, M., Narita, M., Mizoguchi, H., Tseng, L.F., 1995. Inhibition of protein kinase C, but not of protein kinase A, blocks the development of acute antinociceptive tolerance to an intrathecally administered μ-opioid receptor agonist in the mouse. Eur. J. Pharmacol. 280, R1–R3. Narita, M., Mizoguchi, H., Kampine, J.P., Tseng, L.F., 1996. Role of protein kinase C in desensitization of spinal δ-opioid-mediated antinociception in the mouse. Br. J. Pharmacol. 118, 1829–1835. Narita, M., Mizoguchi, H., Kampine, J.P., Tseng, L.F., 1997a. The effect of pretreatment with a δ2-opioid receptor antisense oligodeoxynucleotide on the recovery from acute antinociceptive tolerance to δ2-opioid receptor agonist in the mouse spinal cord. Br. J. Pharmacol. 120, 587–592. Narita, M., Mizoguchi, H., Nagase, H., Tseng, L.F., 1997b. Use of antisense oligodeoxynucleotide to δ-opioid receptor mRNA in the study of turnover of δ-opioid receptors in the spinal cord of the mouse. Psychopharmacology 133, 347–350. Ogawa, T., Miyamae, T., Murayama, K., Okuyama, K., Okayama, T., Hagiwara, M., Sakurada, S., Morikawa, T., 2002. Synthesis and structure-activity relationships of an orally available and long-acting analgesic peptide, Nα-amidino-Tyr-D-Arg-PheMeβAla-OH (ADAMB). J. Med. Chem. 45, 5081–5089. Pan, Y.X., Xu, J., Bolan, E., Abbadie, C., Chang, A., Zuckerman, A., Rossi, G., Pasternak, G.W., 1999. Identification and characterization of three new alternatively spliced μ-opioid receptor isoforms. Mol. Pharmacol. 56 936-403. Pan, Y.X., Xu, J., Bolan, E., Chang, A., Mahurter, L.A., Rossi, G., Pasternak, G.W., 2000. Isolation and expression of a novel alternatively spliced mu opioid receptor isoform, MOR-1F. FEBS Lett. 466, 337–340.
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Pan, Y.X., Xu, J., Mahurter, L., Bolan, E., Xu, M., Pasternak, G.W., 2001. Generation of the mu opioid receptor (MOR-1) protein by three new splice variants of the Oprm gene. Proc. Natl. Acad. Sci. U.S.A. 98, 14084–14089. Pan, Y.X., Xu, J., Bolan, E., Moskowitz, H.S., Xu, M., Pasternak, G.W., 2005. Identification of four novel exon 5 splice variants of the mouse μ-opioid receptor gene: functional consequences of C-terminal splicing. Mol. Pharmacol. 68, 866–875. Pasternak, G.W., 2004. Multiple opiate receptors: déjà vu all over again. Neuropharmacology 47, 312–323. Sakurada, S., Hayashi, T., Yuhki, M., Orito, T., Zadina, J.E., Kastin, A.J., Fujimura, T., Murayama, K., Sakurada, C., Sakurada, T., Narita, M., Suzuki, T., Tan-no, K., Tseng, L.F., 2001. Differential antinociceptive effects induced by intrathecally administered endomorphin-1 and endomorphin-2 in the mouse. Eur. J. Pharmacol. 427, 203–210. Suh, H.H., Tseng, L.F., 1990. Lack of antinociceptive cross-tolerance between intracerebroventricularly administered β-endorphin and morphine or DPDPE in mice. Life Sci. 46, 759–765.
Szeto, H.H., Soong, Y., Wu, D., Qian, X., Zhao, G.M., 2003. Endogenous opioid peptides contribute to antinociceptive potency of intrathecal [Dmt1]DALDA. J. Pharmacol. Exp. Ther. 305, 696–702. Wang, J.B., Imai, Y., Eppler, C.M., Gregor, P., Spivak, C.E., Uhl, G.R., 1993. μ Opiate receptor: cDNA cloning and expression. Proc. Natl. Acad. Sci. U.S.A. 90, 10230–10234. Wu, H.E., Hung, K.C., Mizoguchi, H., Fujimoto, J.M., Tseng, L.F., 2001. Acute antinociceptive tolerance and asymmetric cross-tolerance between endomorphin-1 and endomorphin-2 given intracerebroventricularly in the mouse. J. Pharmacol. Exp. Ther. 299, 1120–1125. Wu, H.E., Darpolor, M., Nagase, H., Tseng, L.F., 2003. Acute antinociceptive tolerance and partial cross-tolerance to endomorphin-1 and endomorphin-2 given intrathecally in the mouse. Neurosci. Lett. 248, 139–142.
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Neuropharmacology and Analgesia
Involvement of mouse μ-opioid receptor splice variants in the spinal antinociception induced by the dermorphin tetrapeptide analog amidino-TAPA Hirokazu Mizoguchi a, Chizuko Watanabe a, Takayuki Higashiya a, Satoshi Takeda a, Kaori Moriyama a, Akihiko Yonezawa a, Takumi Sato b, Takaaki Komatsu c, Tsukasa Sakurada c, Shinobu Sakurada a,⁎ a b c
Department of Physiology and Anatomy, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan Department of Pharmacology, Nihon Pharmaceutical University, 10281 Komuro, Ina-machi, Kitaadachi-gun, Saitama 362-0806, Japan First Department of Pharmacology, Daiichi College of Pharmaceutical Sciences, 22-1 Tamagawa-cho, Minami-ku, Fukuoka 815-8511, Japan
a r t i c l e
i n f o
Article history: Received 20 February 2010 Received in revised form 2 October 2010 Accepted 16 October 2010 Available online 31 October 2010 Keywords: Antinociception Acute tolerance mu-Opioid receptor Opioid peptides Splice variants Spinal cord
a b s t r a c t The involvement of the mouse μ-opioid receptor (mMOR-1) splice variants in the antinociceptive effect of intrathecally (i.t.) administered Nα-amidino-Tyr-D-Arg-Phe-β-Ala (amidino-TAPA) and [D-Ala2,N-MePhe4, Gly-ol5]enkephalin (DAMGO) was investigated in mice by monitoring the recovery from acute antinociceptive tolerance to amidino-TAPA and DAMGO. A single i.t. pretreatment with DAMGO produced an acute antinociceptive tolerance, which peaked at 2 h and disappeared within 5 h after the pretreatment. In contrast, a single i.t. pretreatment with amidino-TAPA produced an acute antinociceptive tolerance, which disappeared within 3 h after the pretreatment. The concomitant i.t. pretreatment with an antisense oligodeoxynucleotide (ODN) for exon-1, exon-12, exon-13 or exon-14 of mMOR-1 maintained the acute antinociceptive tolerance to amidino-TAPA for 24 h after the pretreatment. On the other hand, the concomitant i.t. pretreatment with an antisense ODN for exon-1 of mMOR-1, but not an antisense ODN for exon-12, exon-13 or exon-14 of mMOR-1, maintained the acute antinociceptive tolerance to DAMGO for 24 h after the pretreatment. The present results suggest that the spinal antinociception of amidino-TAPA is partially mediated through the activation of the amidino-TAPA-sensitive and DAMGO-insensitive mMOR-1 splice variants MOR-1J, MOR-1K and MOR-1L, which contain the sequence encoded by exon-12, exon-13 and exon-14, respectively. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Nα-Amidino-Tyr-D-Arg-Phe-β-Ala (amidino-TAPA) is an N-terminal tetrapeptide derivative of dermorphin. It has a very high affinity and selectivity for μ-opioid receptors, and produces an extremely potent and long-lasting antinociception (Mizoguchi et al., 2007; Ogawa et al., 2002). The antinociceptive potency of amidino-TAPA is 10-fold, 1.2-fold or 650-fold higher than that of morphine after subcutaneous, oral or intrathecal (i.t.) injections, respectively. Unlike the traditional μ-opioid receptor agonist DAMGO, the antinociceptive effect of i.t.-administered amidino-TAPA is mediated through the release of the endogenous κ-opioid peptides dynorphin A, dynorphin B and α-neo-endorphin and the endogenous δ-opioid peptide [Leu5] enkephalin via activation of μ-opioid receptors in the spinal cord (Mizoguchi et al., 2007). The distinct antinociceptive profiles of amidino-TAPA may be mediated by the activation of distinct μ-opioid receptors that are sensitive to amidino-TAPA but insensitive to DAMGO.
⁎ Corresponding author. Tel.: +81 22 727 0124; fax: +81 22 727 0125. E-mail address: [email protected] (S. Sakurada). 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.10.049
Since the initial cloning of the μ-opioid receptor gene MOR-1 (Chen et al., 1993; Wang et al., 1993), 33 splice variants have been identified in mouse MOR-1 (mMOR-1) mRNA (Doyle et al., 2007a,b; Kvam et al., 2004; Pan et al., 2001, 2005). The sequence variability of the splice variants is found in the sequences of the N-terminal and first transmembrane region, and in the sequence of the intracellular Cterminal region. The selectivities and intrinsic activities of various μopioid receptor agonists for 6 of the splice variants (MOR-1, 1A, 1C, 1D, 1E, and 1F), that contain the sequences encoded by exon-1, have been described in CHO cells over-expressing each of these splice variants (Pasternak, 2004). However, no remarkable differences in the selectivity and intrinsic activity of any μ-opioid receptor agonist were observed among the splice variants examined. For the other splice variants, the selectivity and intrinsic activity of μ-opioid receptor agonists is still unknown. Although the distribution of most of the splice variants in the rodent central nervous system have been described (Doyle et al., 2007a,b; Pan et al., 1999, 2000, 2001, 2005), the physiological roles of each splice variant are still unknown. A single intracerebroventricular or i.t. injection of opioid receptor agonists induces a rapid and short-term reduction of their antinociceptive effect, the so-called “acute antinociceptive tolerance” (Narita et al., 1995, 1996; Suh and Tseng, 1990; Wu et al., 2001, 2003). The development of acute antinociceptive tolerance is considered to
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be a receptor-dependent phenomenon. We have previously reported that the acute antinociceptive tolerance to a δ-opioid receptor agonist is caused by the internalization and degradation of the δ-opioid receptor following the single treatment with a δ-opioid receptor agonist (Narita et al., 1996, 1997a,b). The recovery from the acute antinociceptive tolerance to a δ-opioid receptor agonist is produced by newly synthesized δ-opioid receptors, which are associated with the natural turnover of δ-opioid receptors. Therefore, the suppression of the synthesis of new δ-opioid receptors can maintain the acute antinociceptive tolerance to an δ-opioid receptor agonist. The present study was conducted to identify the mMOR-1 splice variants involved in the spinal antinociceptive effect of amidino-TAPA, but not DAMGO, by monitoring the recovery from acute antinociceptive tolerance to amidino-TAPA and DAMGO.
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following sequences: 5’-GCC CAC TAC ACA CAC GAT AGA-3’ (antisense ODN for exon-1), 5’-GTG AAG CAC TGT CTT CTA AAG GGG-3’ (antisense ODN for exon-12), 5’-TCA GTC TTT ATC AGC TCA CCG CCA-3’ (antisense ODN for exon-13) and 5’-TAT TTT GAT TTT CAG TTG ATT C-3’ (antisense ODN for exon-14). 2.5. Statistical analyses The statistical significance of differences between the groups was assessed with a one-way analysis of variance (ANOVA) and a two-way ANOVA followed by Dunnett's test and Bonferroni's test, respectively. 3. Results 3.1. Acute antinociceptive tolerance to DAMGO and amidino-TAPA
2. Materials and methods All experiments were performed following the approval of the Ethics Committee for Animal Experiments at Tohoku Pharmaceutical University and according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Every effort was made to minimize the number and any suffering of the animal used in the following experiments. 2.1. Animals Male ddY mice (Japan SLC, Hamamatsu, Japan) weighing 22–25 g were used. The animals were housed in a room maintained at 22– 23 °C and 50–60% relative humidity with an alternating 12-h light/ dark cycle. Food and water were available ad libitum. Mice were used only once. 2.2. Assessment of antinociception Antinociception was determined with the tail-flick test (D'Amour and Smith, 1941). For measurement of the latency of the tail-flick response, mice were gently held by hand with their tail positioned in an apparatus (Ugo Basile, Italy) for radiant heat stimulation on the ventral surface of the tail. The intensity of the heat stimulus was adjusted so that the animal withdrew its tail after 2.5–3.5 s. The inhibition of the tail-flick response was expressed as the percent maximal possible effect, % MPE, which was calculated as follows: [(T1 − T0)/(T2 − T0)]× 100, where T0 and T1 are the tail-flick latencies before and after the treatments, respectively, and T2 is the cutoff time, set at 10 s to avoid injury to the tail. The antinociceptive effect was represented as the mean ± S.E.M. for at least 10 mice. The dose–response curves and ED50 values with their 95% confidence intervals were calculated with a computer-assisted curve-fitting program (GraphPad Prism; GraphPad Software, Inc., San Diego, CA).
Groups of mice were injected i.t. with DAMGO (10 pmol), amidino-TAPA (2 pmol) or aCSF, and the antinociceptive effects induced by DAMGO and amidino-TAPA were measured. The i.t. administration of DAMGO or amidino-TAPA resulted in a marked antinociception with approximately equal efficacy (87.54% and 85.38% MPE for DAMGO and amidino-TAPA, respectively, at their peak-effect time) at the doses used (Fig. 1). The antinociceptive effect of amidino-TAPA developed rapidly, reached its maximal effect at 10 min after the treatment, and then disappeared slowly by 120 min. A more prolonged antinociceptive effect was observed for amidinoTAPA than for DAMGO, whose antinociceptive effect peaked at 10 min and disappeared by 45 min after the treatment. In the following experiments, to identify the acute antinociceptive tolerance to DAMGO or amidino-TAPA, the antinociceptive effects of i.t.-administered DAMGO (10 pmol) or amidino-TAPA (2 pmol) were measured 10 min after the treatment in mice receiving the various pretreatments. Groups of mice were pretreated i.t. with aCSF or DAMGO (10 pmol) 1, 2, 3, 4, 5, or 6 h before the i.t. treatment with DAMGO (10 pmol), and the antinociception induced by DAMGO was measured 10 min after the treatment. The mice pretreated with DAMGO 1 h before showed an antinociceptive effect of DAMGO treatment that was equipotent to mice pretreated with aCSF (Fig. 2A). However, in mice pretreated with DAMGO 2 h before, the antinociceptive effect of DAMGO was dramatically suppressed compared with mice pretreated
2.3. Intrathecal injection The i.t. injection was performed following the method described by Hylden and Wilcox (1980) using a 29-gauge stainless-steel needle attached to a 50-μl Hamilton microsyringe. For i.t. injections, drugs were dissolved in sterile artificial cerebrospinal fluid (aCSF) containing 126.6 mM NaCl, 2.5 mM KCl, 2.0 mM MgCl2, and 1.3 mM CaCl2. The volume for the i.t. injections was 5 μl. 2.4. Drugs The drugs used were amidino-TAPA (Peptides Institute, Osaka, Japan) and DAMGO (Sigma-Aldrich, St. Louis, MO). Antisense phosphorothioate-linked oligodeoxynucleotides (ODNs) for exon-1, exon-12, exon-13, and exon-14 of mMOR-1, which were purchased from The Midland Certified Reagent Company (Midland, TX), had the
Fig. 1. Antinociceptive effects of DAMGO and amidino-TAPA in the ddY mouse spinal cord. Groups of mice were injected i.t. with DAMGO (10 pmol) or amidino-TAPA (2 pmol), and the tail-flick inhibition induced by DAMGO or amidino-TAPA was measured for 90 or 180 min, respectively. The antinociceptive effect was calculated as the mean ± S.E.M for at least 10 mice. The statistical significance of the differences between the groups was assessed with a two-way ANOVA followed by Bonferroni's test. The F values of the two-way ANOVA for DAMGO and amidino-TAPA against aCSF were F[1,126] = 134.7 (P b 0.001) and F[1,180] = 340.4 (P b 0.001), respectively. ***P b 0.001 vs. the aCSF-treated group.
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antinociceptive tolerance to amidino-TAPA 3 h after the pretreatment was determined under conditions where the new synthesis of most of the mMOR-1 splice variants had been suppressed by the pretreatment with an antisense ODN for exon-1 of mMOR-1. Groups of mice pretreated i.t. with aCSF or an antisense ODN for exon-1 of mMOR-1 (5 μg) 24 h before, were injected i.t. with aCSF or amidino-TAPA (2 pmol) 3 h before the i.t. treatment with amidino-TAPA (2 pmol), and the tail-flick inhibition induced by amidino-TAPA was measured at 10 min after the treatments. Like the data presented in Fig. 2B, no suppression of amidino-TAPA-induced antinociception at 3 h after the pretreatment with amidino-TAPA (acute antinociceptive tolerance to amidino-TAPA) was observed in mice pretreated with aCSF 24 h before (data not shown). However, in mice pretreated with an antisense ODN for exon-1 of mMOR-1 24 h before, a marked suppression of amidino-TAPA-induced antinociception at 3 h after the pretreatment with amidino-TAPA (the acute antinociceptive tolerance to amidino-TAPA) was observed.
3.2. Retention of the acute antinociceptive tolerance to DAMGO or amidino-TAPA by co-pretreatment with exon-specific antisense ODNs for mMOR-1
Fig. 2. Acute antinociceptive tolerance to DAMGO and amidino-TAPA in the ddY mouse spinal cord. (A) Groups of mice were pretreated i.t. with aCSF or DAMGO (10 pmol) 1, 2, 3, 4, 5, or 6 h before the i.t. treatment with DAMGO (10 pmol), and the antinociception induced by DAMGO was measured 10 min after the treatment. (B) Groups of mice were pretreated i.t. with aCSF or amidino-TAPA (2 pmol) 3, 4, 5, 6, 7, or 9 h before the i.t. treatment with amidino-TAPA (2 pmol), and the antinociception induced by amidinoTAPA was measured 10 min after the treatment. The antinociceptive effect was calculated as the mean ± S.E.M for at least 10 mice. The statistical significance of the differences between the groups was assessed with a two-way ANOVA followed by Bonferroni's test. (A) The F value of the two-way ANOVA was F[1,124] = 21.90 (P b 0.001). *P b 0.05, ***P b 0.001 vs. the aCSF-pretreated group. (B) The F value of the two-way ANOVA was F[1,117] = 0.3782 (P = 0.5397).
with aCSF. The suppression of DAMGO-induced antinociception (acute antinociceptive tolerance to DAMGO) gradually disappeared. At 5 h after the pretreatment with DAMGO, the antinociceptive effect of DAMGO treatment had completely recovered to the same magnitude observed in mice pretreated with aCSF. Other groups of mice were pretreated i.t. with aCSF or amidinoTAPA (2 pmol) 3, 4, 5, 6, 7, or 9 h before the i.t. treatment with amidino-TAPA (2 pmol), and the antinociception induced by amidinoTAPA was measured 10 min after the treatment. In contrast to DAMGO, the antinociceptive effect of amidino-TAPA in mice pretreated with amidino-TAPA was exactly equipotent with that in mice pretreated with aCSF at all time points measured after the pretreatment (Fig. 2B). In other words, no suppression of amidinoTAPA-induced antinociception after the pretreatment with amidinoTAPA (acute antinociceptive tolerance to amidino-TAPA) was observed at any time point tested. Together with the evidence that the antinociceptive effect of amidino-TAPA is long-lasting and it takes 2 h to return the nociceptive threshold to the baseline level after the treatment, the present results suggests the possibility that the acute antinociceptive tolerance to amidino-TAPA develops rapidly and disappears within 3 h. To investigate this possibility, the acute
The mMOR-1 splice variants involved in the acute antinociceptive tolerance to DAMGO or amidino-TAPA in the mouse spinal cord were determined using the exon-specific antisense ODNs for mMOR-1. Groups of mice were pretreated i.t. with aCSF, an antisense ODN for exon-1 of mMOR-1 (5 μg), μ-opioid receptor agonists (10 pmol of DAMGO or 2 pmol of amidino-TAPA), or μ-opioid receptor agonists in combination with an antisense ODN for exon-1 of mMOR-1 24 h before the i.t. treatment with μ-opioid receptor agonists (10 pmol of DAMGO or 2 pmol of amidino-TAPA), and the tail-flick inhibition induced by μ-opioid receptor agonists was measured at 10 min after the treatments. The pretreatment with an antisense ODN for exon-1 of mMOR-1 24 h before did not alter the antinociception induced by either DAMGO or amidino-TAPA (Fig. 3). The antinociception induced by DAMGO and amidino-TAPA also was not affected by the 24h pretreatment with DAMGO and amidino-TAPA, respectively. The acute antinociceptive tolerance to DAMGO and amidino-TAPA was not observed 24 h after the pretreatment with DAMGO and amidinoTAPA, respectively. In contrast, the antinociception induced by DAMGO or amidino-TAPA was significantly attenuated by the concomitant pretreatment with the antisense ODN for exon-1 of mMOR-1 and either DAMGO or amidino-TAPA, respectively. The acute antinociceptive tolerance to DAMGO or amidino-TAPA was still maintained for 24 h after the pretreatment by the concomitant pretreatment with the antisense ODN for exon-1 of mMOR-1. The involvement of MOR-1J (splice variant containing the sequence encoded by exon-12), MOR-1K (splice variant containing the sequence encoded by exon-13) and MOR-1L (splice variant containing the sequence encoded by exon-14) in the acute antinociceptive tolerance to DAMGO or amidino-TAPA in the mouse spinal cord was also investigated on the same experimental schedule, using antisense ODNs for exon-12, exon-13 and exon-14 of mMOR-1, respectively. The pretreatment with antisense ODNs for either exon-12, exon-13 or exon-14 of mMOR-1 24 h before did not alter the antinociception induced by either DAMGO or amidino-TAPA (Figs. 4–6). AmidinoTAPA-induced antinociception was significantly attenuated by the concomitant pretreatment with amidino-TAPA and antisense ODNs for either exon-12, exon-13 or exon-14 of mMOR-1 (Figs. 4B, 5B, 6B), whereas DAMGO-induced antinociception was not altered by the concomitant pretreatment with DAMGO and antisense ODNs for either exon-12, exon-13 or exon-14 of mMOR-1 (Figs. 4A, 5A, 6A). The acute antinociceptive tolerance to amidino-TAPA, but not to DAMGO, was maintained for 24 h after the pretreatment by antisense ODNs for either exon-12, exon-13 or exon-14 of mMOR-1.
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Fig. 3. Effects of an antisense ODN for exon-1 of mMOR-1 on the acute antinociceptive tolerance to DAMGO and amidino-TAPA in the ddY mouse spinal cord. (A) Groups of mice were pretreated i.t. with aCSF, an antisense ODN for exon-1 of mMOR-1 (AS/Exon1: 5 μg), DAMGO (10 pmol) or DAMGO in combination with AS/Exon-1 24 h before the i.t. treatment with DAMGO (10 pmol), and the tail-flick inhibition induced by DAMGO was measured at 10 min after the treatments. (B) Groups of mice were pretreated i.t. with aCSF, AS/Exon-1 (5 μg), amidino-TAPA (2 pmol) or amidino-TAPA in combination with AS/Exon-1 24 h before the i.t. treatment with amidino-TAPA (2 pmol), and the tail-flick inhibition induced by amidino-TAPA was measured at 10 min after the treatments. The antinociceptive effect was calculated as the mean ± S.E.M for at least 10 mice. The statistical significance of the differences between the groups was assessed with a one-way ANOVA followed by Dunnett's test. (A) The F value of the one-way ANOVA was F[3,35] = 8.836 (P b 0.001). **P b 0.01 vs. the aCSF-pretreated group. (B) The F value of the one-way ANOVA was F[3,36] = 4.271 (P b 0.05). *P b 0.05 vs. the aCSFpretreated group.
4. Discussion Amidino-TAPA has a very high affinity and selectivity for μ-opioid receptors, and shows an extremely potent and long-lasting antinociception that is mediated by μ-opioid receptors (Mizoguchi et al., 2007; Ogawa et al., 2002). Unlike the traditional μ-opioid receptor agonist DAMGO, amidino-TAPA has a distinct spinal antinociceptive mechanism that is mediated by the release of the endogenous δ-opioid peptide [Leu5]enkephalin and the endogenous κ-opioid peptides dynorphin A, dynorphin B and α-neo-endorphin, which is initiated by the activation of spinal μ-opioid receptors (Mizoguchi et al., 2007). An antinociception that is mediated through the release of endogenous opioid peptides in the spinal cord is also observed for the other selective μ-opioid receptor agonists endomorphin-2 (Mizoguchi et al., 2006b; Sakurada et al., 2001), Tyr-D-Arg-Phe-Sar (TAPS: Mizoguchi et al., 2006a) and dimethyl-Tyr-D-Arg-Phe-Lys-NH2
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Fig. 4. Effects of an antisense ODN for exon-12 of mMOR-1 on the acute antinociceptive tolerance to DAMGO and amidino-TAPA in the ddY mouse spinal cord. (A) Groups of mice were pretreated i.t. with aCSF, an antisense ODN for exon-12 of mMOR-1 (AS/ Exon-12: 5 μg), DAMGO (10 pmol) or DAMGO in combination with AS/Exon-12 24 h before the i.t. treatment with DAMGO (10 pmol), and the tail-flick inhibition induced by DAMGO was measured at 10 min after the treatments. (B) Groups of mice were pretreated i.t. with aCSF, AS/Exon-12 (5 μg), amidino-TAPA (2 pmol) or amidino-TAPA in combination with AS/Exon-12 24 h before the i.t. treatment with amidino-TAPA (2 pmol), and the tail-flick inhibition induced by amidino-TAPA was measured at 10 min after the treatments. The antinociceptive effect was calculated as the mean± S.E.M for at least 10 mice. The statistical significance of the differences between the groups was assessed with a one-way ANOVA followed by Dunnett's test. (A) The F value of the one-way ANOVA was F[3,36]= 0.1393 (P= 0.9358). (B) The F value of the one-way ANOVA was F [3,76]= 3.444 (P b 0.05). *P b 0.05 vs. the aCSF-pretreated group.
([Dmt1]DALDA: Szeto et al., 2003). The evidence clearly suggests that these selective μ-opioid receptor agonists stimulate different μ-opioid receptors than DAMGO to produce the antinociception. At present, 33 splice variants for mMOR-1 have been identified (Doyle et al., 2007a, b; Kvam et al., 2004; Pan et al., 2001, 2005). The release of endogenous opioid peptides by the above μ-opioid receptor agonists may be mediated by the activation of specific splice variants of mMOR-1 that are insensitive to DAMGO. In the present study, the mMOR-1 splice variants involved in the spinal antinociception of amidino-TAPA, but not DAMGO, were identified by monitoring the recovery from the acute antinociceptive tolerance to amidino-TAPA. Acute antinociceptive tolerance is the rapid and temporary reduction of the antinociceptive effect of opioid receptor agonists observed after a single pretreatment with the same agonist. In the present study, the pretreatment with DAMGO caused the reduction of its antinociceptive effect (acute antinociceptive tolerance), which peaked at 2 h and disappeared by 5 h after the pretreatment. The
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Fig. 5. Effects of an antisense ODN for exon-13 of mMOR-1 on the acute antinociceptive tolerance to DAMGO and amidino-TAPA in the ddY mouse spinal cord. (A) Groups of mice were pretreated i.t. with aCSF, an antisense ODN for exon-13 of mMOR-1 (AS/ Exon-13: 5 μg), DAMGO (10 pmol) or DAMGO in combination with AS/Exon-13 24 h before the i.t. treatment with DAMGO (10 pmol), and the tail-flick inhibition induced by DAMGO was measured at 10 min after the treatments. (B) Groups of mice were pretreated i.t. with aCSF, AS/Exon-13 (5 μg), amidino-TAPA (2 pmol) or amidino-TAPA in combination with AS/Exon-13 24 h before the i.t. treatment with amidino-TAPA (2 pmol), and the tail-flick inhibition induced by amidino-TAPA was measured at 10 min after the treatments. The antinociceptive effect was calculated as the mean± S.E.M for at least 10 mice. The statistical significance of the differences between the groups was assessed with a one-way ANOVA followed by Dunnett's test. (A) The F value of the one-way ANOVA was F[3,36]=0.4406 (P= 0.7253). (B) The F value of the one-way ANOVA was F [3,36]=5.065 (Pb 0.01). **P b 0.01 vs. the aCSF-pretreated group.
duration of acute antinociceptive tolerance to opioid receptor agonists is variable depending on the subtype of the opioid receptors. Unlike the μ-opioid receptor agonist DAMGO, the acute antinociceptive tolerance to the δ-opioid receptor agonist [D-Ala2]deltorphin II and the κ-opioid receptor agonist U-50,488H both peaked at 3–6 h, but disappeared by 12 h and 24 h after the pretreatment, respectively (Narita et al., 1997a; unpublished observation). The development of acute antinociceptive tolerance to δ-opioid receptor agonist is caused by the internalization and degradation of δ-opioid receptors, and the recovery from the acute antinociceptive tolerance is produced by the newly synthesized δ-opioid receptors (Narita et al., 1997a). The duration of the acute antinociceptive tolerance to opioid receptor agonists may depend on the turnover speed of the opioid receptor, which is identical for each opioid receptor subtype. In the present study, the pretreatment with amidino-TAPA did not show any reduction of its antinociceptive effect (acute antinociceptive tolerance) at 3–9 h after the pretreatment. The antinociceptive effect of amidino-TAPA is long-lasting and it takes 2 h to completely return the
Fig. 6. Effects of an antisense ODN for exon-14 of mMOR-1 on the acute antinociceptive tolerance to DAMGO and amidino-TAPA in the ddY mouse spinal cord. (A) Groups of mice were pretreated i.t. with aCSF, an antisense ODN for exon-14 of mMOR-1 (AS/ Exon-14: 5 μg), DAMGO (10 pmol) or DAMGO in combination with AS/Exon-14 24 h before the i.t. treatment with DAMGO (10 pmol), and the tail-flick inhibition induced by DAMGO was measured at 10 min after the treatments. (B) Groups of mice were pretreated i.t. with aCSF, AS/Exon-14 (5 μg), amidino-TAPA (2 pmol) or amidino-TAPA in combination with AS/Exon-14 24 h before the i.t. treatment with amidino-TAPA (2 pmol), and the tail-flick inhibition induced by amidino-TAPA was measured at 10 min after the treatments. The antinociceptive effect was calculated as the mean± S.E.M for at least 10 mice. The statistical significance of the differences between the groups was assessed with a one-way ANOVA followed by Dunnett's test. (A) The F value of the one-way ANOVA was F[3,36]= 0.1991 (P= 0.8963). (B) The F value of the one-way ANOVA was F [3,36]= 6.497 (P b 0.01). **P b 0.01 vs. the aCSF-pretreated group.
nociceptive threshold to the baseline level after the treatment. Based on the above evidence, the present results suggest the possibility that the acute antinociceptive tolerance to amidino-TAPA develops rapidly and disappears within 3 h. To investigate this possibility, the acute antinociceptive tolerance to amidino-TAPA at 3 h after the pretreatment was determined under conditions where the new synthesis of most of the mMOR-1 splice variants had been suppressed by the pretreatment with antisense ODN for exon-1 of mMOR-1. Under these conditions, amidino-TAPA showed a marked acute antinociceptive tolerance, suggesting that the acute antinociceptive tolerance to amidino-TAPA developed rapidly and disappeared within 3 h. The suggestion that the duration of acute antinociceptive tolerance to amidino-TAPA is much shorter than that to DAMGO is another piece of evidence to support the hypothesis that amidino-TAPA stimulates different μ-opioid receptors than DAMGO.
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By monitoring the recovery from the acute antinociceptive tolerance to DAMGO and amidino-TAPA, the involvement of mMOR1 splice variants in the acute antinociceptive tolerance to DAMGO or amidino-TAPA was determined using exon-specific antisense ODNs for mMOR-1. In general, the prolonged pretreatment (usually 4 days) with antisense ODNs for opioid receptors is required to desensitize the opioid receptors (Narita et al., 1997b). Because antisense ODNs for the opioid receptor itself do not have any effect on existing opioid receptors on the cell membrane surface, it should take 3–4 days to desensitize existing opioid receptors with endogenous opioid peptides. In fact, a single 24-h pretreatment with an antisense ODN for exon-1 of mMOR-1 did not alter DAMGO-induced antinociception, although a 4-day pretreatment with the same antisense ODN significantly suppressed the DAMGO-induced antinociception (unpublished observation). However, a single 24-h but concomitant pretreatment with an antisense ODN for exon-1 of mMOR-1 and DAMGO suppressed DAMGO-induced antinociception, suggesting that the suppression of the synthesis of new mMOR-1 splice variants containing the sequence encoded by exon-1 can maintain the acute antinociceptive tolerance to DAMGO for 24 h after the pretreatment, which usually disappeared within 5 h after the pretreatment. This is a simple but accurate method for desensitizing a specific subtype of opioid receptor with a single pretreatment. In the present study, the concomitant pretreatment with an antisense ODN for exon-1 of mMOR-1 and amidino-TAPA maintained the acute antinociceptive tolerance to amidino-TAPA for 24 h after the pretreatment, suggesting that the splice variants of mMOR-1 that are desensitized by pretreatment with amidino-TAPA, as well as DAMGO, are the splice variants containing the sequence encoded by exon-1. Most of the splice variants for mMOR-1 identified contain the sequence encoded by exon-1 (Doyle et al., 2007a,b; Kvam et al., 2004; Pan et al., 2001, 2005). Pasternak and his colleagues described the selectivity and intrinsic activity of various μ-opioid receptor agonists for mMOR-1 splice variants containing the sequences encoded by exon-1 (Bolan et al., 2004; Pan et al., 1999, 2000; Pasternak, 2004). However, no remarkable differences in the selectivity and intrinsic activity of any μopioid receptor agonists were observed among the mMOR-1 splice variants examined. Therefore, in the present study, we focused on the other mMOR-1 splice variants, MOR-1J, MOR-1K or MOR-1L, which contain the sequence encoded by exon-12, exon-13 or exon-14, respectively. We found that amidino-TAPA-induced antinociception is significantly suppressed by a 24-h concomitant pretreatment with amidino-TAPA and an antisense ODN for either exon-12, exon-13 or exon-14 of mMOR-1. The suppression of the new synthesis of MOR-1J, MOR-1K or MOR-1L can maintain the acute antinociceptive tolerance to amidino-TAPA for 24 h after the pretreatment, suggesting that the splice variants of mMOR-1 desensitized by pretreatment with amidino-TAPA are MOR-1J, MOR-1K and MOR-1L. In contrast, DAMGO-induced antinociception was not affected by a 24-h concomitant pretreatment with DAMGO and an antisense ODN for either exon-12, exon-13 or exon-14 of mMOR-1, suggesting that MOR-1J, MOR-1K and MOR-1L are not the splice variants desensitized by pretreatment with DAMGO. In other words, these splice variants are amidino-TAPA-sensitive and DAMGO-insensitive mMOR-1 splice variants. At present, the physiological roles of the 33 identified splice variants of mMOR-1 are still unclear. We have previously found that, unlike DAMGO, amidino-TAPA induces the release of the endogenous κ-opioid peptides dynorphin A, dynorphin B and α-neo-endorphin and the endogenous δ-opioid peptide [Leu5]enkephalin in the spinal cord to produce the antinociception. In the present study, we have identified the amidino-TAPA-sensitive and DAMGO-insensitive mMOR-1 splice variants, MOR-1J, MOR-1K and MOR-1L, in the spinal cord. Together with the previous observations, the present results suggest the possibility that the release of endogenous opioid peptides by amidino-TAPA is mediated by the amidino-TAPA-sensitive and
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DAMGO-insensitive mMOR-1 splice variants MOR-1J, MOR-1K and MOR-1L.
5. Conclusion In conclusion, the spinal antinociception of amidino-TAPA is partially mediated through the activation of the distinct mMOR-1 splice variants MOR-1J, MOR-1K or MOR-1L, which contain the sequence encoded by exon-12, exon-13 or exon-14, respectively. These splice variants are amidino-TAPA-sensitive and DAMGOinsensitive mMOR-1 splice variants, which may be responsible for the distinct antinociceptive properties of amidino-TAPA.
Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research (C) [KAKENHI 21600013 and 22600009] from the Japan Society for the Promotion of Science, and a Matching Fund Subsidy for Private Universities from the Ministry of Education, Culture, Sports, Science, and Technology Japan (2010–2014).
References Bolan, E.A., Pan, Y.X., Pasternak, G.W., 2004. Functional analysis of MOR-1 splice variants of the mouse mu opioid receptor gene Oprm. Synapse 51, 11–18. Chen, Y., Mestek, A., Liu, J., Hurley, A., Yu, L., 1993. Molecular cloning and functional expression of a μ-opioid receptor from rat brain. Mol. Pharmacol. 44, 8–12. D'Amour, F.E., Smith, D.L., 1941. A method for determining loss of pain sensation. J. Pharmacol. Exp. Ther. 72, 74–79. Doyle, G.A., Sheng, X.R., Lin, S.S., Press, D.M., Grice, D.E., Buono, R.J., Ferraro, T.N., Berrettini, W.H., 2007a. Identification of three mouse μ-opioid receptor (MOR) gene (Oprm1) splice variants containing a newly identified alternatively spliced exon. Gene 388, 135–147. Doyle, G.A., Sheng, X.R., Lin, S.S., Press, D.M., Grice, D.E., Buono, R.J., Ferraro, T.N., Berrettini, W.H., 2007b. Identification of five mouse μ-opioid receptor (MOR) gene (Oprm1) splice variants containing a newly identified alternatively spliced exon. Gene 395, 98–107. Hylden, J.L., Wilcox, G.L., 1980. Intrathecal morphine in mice: a new technique. Eur. J. Pharmacol. 67, 313–316. Kvam, T.M., Baar, C., Rakvåg, T.T., Kaasa, S., Krokan, H.E., Skorpen, F., 2004. Genetic analysis of the murine μ opioid receptor: increased complexity of Oprm gene splicing. J. Mol. Med. 82, 250–255. Mizoguchi, H., Ito, K., Watanabe, H., Watanabe, C., Katsuyama, S., Fujimura, T., Sakurada, T., Sakurada, S., 2006a. Contribution of spinal μ1-opioid receptors and dynorphin B to the antinociception induced by Tyr-D-Arg-Phe-Sar. Peptides 27, 2786–2793. Mizoguchi, H., Watanabe, H., Hayashi, T., Sakurada, W., Sawai, T., Fujimura, T., Sakurada, T., Sakurada, S., 2006b. Possible involvement of dynorphin A (1–17) release via μ1opioid receptors in spinal antinociception by endomorphin-2. J. Pharmacol. Exp. Ther. 317, 362–368. Mizoguchi, H., Watanabe, C., Watanabe, H., Moriyama, K., Sato, B., Ohwada, K., Yonezawa, A., Sakurada, T., Sakurada, S., 2007. Involvement of endogenous opioid peptides in the antinociception induced by the novel dermorphin tetrapeptide analog amidino-TAPA. Eur. J. Pharmacol. 560, 150–159. Narita, M., Narita, M., Mizoguchi, H., Tseng, L.F., 1995. Inhibition of protein kinase C, but not of protein kinase A, blocks the development of acute antinociceptive tolerance to an intrathecally administered μ-opioid receptor agonist in the mouse. Eur. J. Pharmacol. 280, R1–R3. Narita, M., Mizoguchi, H., Kampine, J.P., Tseng, L.F., 1996. Role of protein kinase C in desensitization of spinal δ-opioid-mediated antinociception in the mouse. Br. J. Pharmacol. 118, 1829–1835. Narita, M., Mizoguchi, H., Kampine, J.P., Tseng, L.F., 1997a. The effect of pretreatment with a δ2-opioid receptor antisense oligodeoxynucleotide on the recovery from acute antinociceptive tolerance to δ2-opioid receptor agonist in the mouse spinal cord. Br. J. Pharmacol. 120, 587–592. Narita, M., Mizoguchi, H., Nagase, H., Tseng, L.F., 1997b. Use of antisense oligodeoxynucleotide to δ-opioid receptor mRNA in the study of turnover of δ-opioid receptors in the spinal cord of the mouse. Psychopharmacology 133, 347–350. Ogawa, T., Miyamae, T., Murayama, K., Okuyama, K., Okayama, T., Hagiwara, M., Sakurada, S., Morikawa, T., 2002. Synthesis and structure-activity relationships of an orally available and long-acting analgesic peptide, Nα-amidino-Tyr-D-Arg-PheMeβAla-OH (ADAMB). J. Med. Chem. 45, 5081–5089. Pan, Y.X., Xu, J., Bolan, E., Abbadie, C., Chang, A., Zuckerman, A., Rossi, G., Pasternak, G.W., 1999. Identification and characterization of three new alternatively spliced μ-opioid receptor isoforms. Mol. Pharmacol. 56 936-403. Pan, Y.X., Xu, J., Bolan, E., Chang, A., Mahurter, L.A., Rossi, G., Pasternak, G.W., 2000. Isolation and expression of a novel alternatively spliced mu opioid receptor isoform, MOR-1F. FEBS Lett. 466, 337–340.
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Pan, Y.X., Xu, J., Mahurter, L., Bolan, E., Xu, M., Pasternak, G.W., 2001. Generation of the mu opioid receptor (MOR-1) protein by three new splice variants of the Oprm gene. Proc. Natl. Acad. Sci. U.S.A. 98, 14084–14089. Pan, Y.X., Xu, J., Bolan, E., Moskowitz, H.S., Xu, M., Pasternak, G.W., 2005. Identification of four novel exon 5 splice variants of the mouse μ-opioid receptor gene: functional consequences of C-terminal splicing. Mol. Pharmacol. 68, 866–875. Pasternak, G.W., 2004. Multiple opiate receptors: déjà vu all over again. Neuropharmacology 47, 312–323. Sakurada, S., Hayashi, T., Yuhki, M., Orito, T., Zadina, J.E., Kastin, A.J., Fujimura, T., Murayama, K., Sakurada, C., Sakurada, T., Narita, M., Suzuki, T., Tan-no, K., Tseng, L.F., 2001. Differential antinociceptive effects induced by intrathecally administered endomorphin-1 and endomorphin-2 in the mouse. Eur. J. Pharmacol. 427, 203–210. Suh, H.H., Tseng, L.F., 1990. Lack of antinociceptive cross-tolerance between intracerebroventricularly administered β-endorphin and morphine or DPDPE in mice. Life Sci. 46, 759–765.
Szeto, H.H., Soong, Y., Wu, D., Qian, X., Zhao, G.M., 2003. Endogenous opioid peptides contribute to antinociceptive potency of intrathecal [Dmt1]DALDA. J. Pharmacol. Exp. Ther. 305, 696–702. Wang, J.B., Imai, Y., Eppler, C.M., Gregor, P., Spivak, C.E., Uhl, G.R., 1993. μ Opiate receptor: cDNA cloning and expression. Proc. Natl. Acad. Sci. U.S.A. 90, 10230–10234. Wu, H.E., Hung, K.C., Mizoguchi, H., Fujimoto, J.M., Tseng, L.F., 2001. Acute antinociceptive tolerance and asymmetric cross-tolerance between endomorphin-1 and endomorphin-2 given intracerebroventricularly in the mouse. J. Pharmacol. Exp. Ther. 299, 1120–1125. Wu, H.E., Darpolor, M., Nagase, H., Tseng, L.F., 2003. Acute antinociceptive tolerance and partial cross-tolerance to endomorphin-1 and endomorphin-2 given intrathecally in the mouse. Neurosci. Lett. 248, 139–142.