SYNAPSE 69:166–171 (2015)
Effects Of (1)-Pentazocine on the Antinociceptive Effects of (–)-Pentazocine in Mice TOMOHISA MORI, JUNPEI OHYA, TOSHIMASA ITOH, YUYA ISE, MASAHIRO SHIBASAKI, AND TSUTOMU SUZUKI* Department of Toxicology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, Tokyo, Japan
KEY WORDS
pentazocine; l-opioid receptor; sigma-1 receptor; antinociception
ABSTRACT Previous studies have shown that sigma-1 receptor chaperone (Sig1R) ligands can regulate pain-related behaviors, and Sig-1R itself can regulate lopioid receptor functions as well as signal transduction. Even though (6)-pentazocine has been used clinically for the treatment of pain through opioid receptors, (1)-pentazocine is known to be a selective Sig-1R agonist. To the best of our knowledge, there is no information available regarding the involvement of Sig-1R agonistic action in the antinociceptive effects of (6)-pentazocine. Therefore, the present study was designed to investigate the effects of (1)-pentazocine on the antinociceptive effects of (–)-pentazocine in mice. Both and (–)-pentazocine induced biphasic antinociceptive effects as measured by the warm-plate test. The early phase, but not the delayed phase, of the antinociceptive effects induced by (–)-pentazocine, which are mediated by the activation of l-opioid receptors, were suppressed by pretreatment with (1)-pentazocine. These results suggest that the innate antinociceptive action of (6)-pentazocine could be marginally reduced by the effects of (1)-pentazocine, but (1)-pentazocine can suppress the antinociceptive effects of (–)-pentazocine at certain time points. Synapse 69:166–171, 2015. VC 2015 Wiley Periodicals, Inc. INTRODUCTION Sigma-1 receptor (Sig-1R) was originally considered to be an opioid receptor based on its displacement of the SKF10047 binding site, and thus far it has been defined as a non-opioid binding site, unlike l-, d-, and j-opioid receptors (Su and Hayashi, 2003). Over the past few years, our understanding of Sig-1R receptors has been changing; Sig-1R is mainly localized at one of the endoplasmic reticulum (ER) domains, and is a ligand-operated ER-chaperone protein that regulates ER stress to promote the proper folding of newly synthesized or unfolded protein (Hayashi and Su, 2007). Furthermore, recent findings have demonstrated that Sig-1R can associate with G-protein coupled receptors or channels, and then regulate the signaling cascade (Cormaci et al., 2007; Kim et al., 2010; Kourrich et al., 2013; Navarro et al., 2010; Su et al., 2009). A large and growing body of evidence has demonstrated that Sig-1Rs agonists exert several pharmacological effects, such as nociceptive, hallucinogenic, neuroprotective and anti-depressant effects. However, the underlying mechanisms by which Sig-1R agonist induces pharmacological effects is still unclear. Although pentazocine has enantiomers, called (1)pentazocine and (–)-pentazocine, the racemic comÓ 2015 WILEY PERIODICALS, INC.
pound (6)-pentazocine has been used for the management of mild to moderate pain in humans. (–)Pentazocine exerts its antinociceptive effects mainly mediated by the activation of opioid receptors (Gutstein and Akil, 2001). Most j-opioid receptor agonists induce dysphoric and psychotomimetic effects, and a high dose of (6)-pentazocine also elicits dysphoric and psychotomimetic effects (Gutstein and Akil, 2001). On the other hand, (1) pentazocine is known to be a selective Sig-1R agonist. The prototypical Sig1R agonist SKF10047 induces hallucinogenic/psychotomimetic effects, and the endogenous hallucinogenic amine N,N-dimethyltryptamine was recently identified as an endogenous sigma-receptor ligand (Fontanilla et al., 2009). Thus, the (1)-pentazocine component of (6)-pentazocine may have a negative Contract grant sponsor: Ministry of Education, Culture, Sports, Science and Technology of Japan; Contract grant sponsor: JSPS KAKENHI; Contract grant number: 23590651. *Correspondence to: Tsutomu Suzuki, Department of Toxicology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan. E-mail: [email protected] Received 29 August 2014; Revised 20 November 2014; Accepted 27 November 2014 DOI: 10.1002/syn.21799 Published online 6 January 2015 in Wiley Online Library (wileyonlinelibrary. com).
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influence on the pharmacological profile of (–)pentazocine. Previous studies have shown that Sig-1R antagonist could enhance opioid receptor agonist-induced antinociceptive effects (Marrazzo et al., 2006; Mei and Pasternak, 2007). Conversely, Sig-1R agonists could have compromising effects on opioid receptor agonist-induced antinociceptive effects under certain conditions (Entrena et al., 2009; Ohsawa et al., 2010; Tseng et al., 2011). However, little or no information is available regarding how (1)-pentazocine affects the pharmacological, and especially the antinociceptive effects, of (–)-pentazocine. Therefore, the present study was designed to investigate the effects of (1)pentazocine on the antinociceptive effects of (-)-pentazocine using warm-plate tests. MATERIALS AND METHODS Animals Male ddY mice (Tokyo Laboratory Animals Science Co. Ltd, Tokyo, Japan), weighing 25–30 g, were housed in groups of 10 in a temperature-controlled (23 6 1 C) specific pathogen free room. The animals were maintained on a 12-h light/dark cycle (lights on 8:00 a.m. to 8:00 p.m.) with laboratory chow and tap water available ad libitum. This study was conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals, Hoshi University School of Pharmacy and Pharmaceutical Sciences, as adopted by the Committee on Animal Research of Hoshi University, which is accredited by the Ministry of Education, Science, Sports and Culture of Japan. Warm-plate test The antinociceptive response was evaluated by recording the latency to paw licking or tapping in the warm-plate test (51 6 0.5 C; Muromachi Kikai, Tokyo, Japan), as described previously (Suzuki et al., 1991). To prevent tissue damage, we established a 30 sec cut-off time. The test was performed after treatment with pentazocine. Each animal served as its own control, and the latency to response was measured both before and after drug administration. In the combination study, (1)-pentazocine was pretreated 15 min before the administration of (–)-pentazocine. NE-100 was also pretreated 15 min before the administration of (1)-pentazocine. Antinociception was calculated as the percentage of the maximum possible effect (% antinociception) according to the following formula: % antinociception 5 (test latency – pre-drug latency)/ (cut-off time – pre-drug latency) 3 100. Cell cultures and treatments Chinese hamster ovary (CHO) cells, which have endogenous Sig-1R, but not l-opioid receptor, were maintained in 75-cm2 flasks (Falcon; BD Biosciences
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Discovery Labware, Bedford, MA) in 10% FBScontaining Minimum Essential Medium Alpha medium at 37 C with 5% CO2 under saturated humidity. The medium was changed every 1–2 days. For drug treatments, cells seeded in 6-well plates were incubated with drugs dissolved in distilled H2O. Controls received the same amount of the vehicle. For transfection, the vector (2–3 lg per well)/Lipofectamine-2000 (6 ll per well) (Invitrogen, Carlsbad, CA) mixture was applied to CHO cells in 6-well plates. After incubation for 4–5 h, the medium was replaced. Western blotting After treatment with drugs, cells were placed on ice, rapidly washed once with ice-cold phosphate-buffered saline (PBS) and harvested in PBS with a rubber policeman. Cell suspensions were centrifuged at 800g for 10 min at 4 C. The supernatants were discarded and the pellets were dissolved with lysis buffer (10 mM Tris-HCl, pH 5 7.5, 150 mM NaCl, 0.5 % Triton X-100, supplemented with a protease inhibitor (Roche Diagnostics, Tokyo, Japan)). Cell lysates were cleared by centrifugation at 20000g at 4 C for 10 min. After deglycosylation with N-glycosidase F (Roche Diagnostics, Tokyo, Japan), cell lysates were denatured by 53 Laemmli sample buffer [250 mM Tris-HCl, pH 5 6.8, 20% glycerol, 10% sodium dodecyl sulfate (SDS)] with b-mercaptoethanol and bromophenol blue overnight at 4 C. Proteins (15–100 lg) were resolved by polyacrylamide SDS-PAGE. Gels were electroblotted onto PVDF membranes (GE Healthcare, Tokyo, Japan) in Towbeen buffer (25 mM Tris-base, 192 mM glycine). Membranes were blocked with 0.5% non-fat dry milk (Bio-Rad) in Tris-buffered saline plus 0.05% Tween20 (TBS-T) for 1h at room temperature. Membranes were incubated overnight at 4 C with an antibody in TBS-T, and probed with the secondary anti-rabbit or anti-mouse immunoglobulin G conjugated with horseradish peroxidase for 1.5 h at room temperature (1:10000 dilution). Protein bands were visualized with a SuperSignal West Femt reagent (Thermo Fisher Scientific K.K., Kanagawa, Japan) with a Fluor Chem 3 system (Laboratory and Medical Supplies, Tokyo, Japan). Anti-FLAG (Sigma-Aldrich Japan K.K., Tokyo, Japan) and anti-HA (Cell Signaling Technology, Tokyo, Japan) were purchased. Coimmunoprecipitation For Sigma-1 receptor-FLAG immunoprecipitation, cell pellets were lysed for 30 min in 150 ll of lysis buffer [10 mM Tris-HCl, pH 5 7.5, 150 mM NaCl, 0.5% Triton X-100, supplemented with a protease inhibitor (Roche Diagnostics, Tokyo, Japan)]. Cell lysates were cleared by centrifugation at 20000g at 4 C for 10 min. Fifteen micro liters of agarosecoupled anti-FLAGM2 antibody (Sigma-Aldrich Japan Synapse
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a volume of 10 ml/kg. Pentazocines were dissolved in water containing 30% lactate acid and 20% saline. Data analysis Data are expressed as the mean 6 S.E.M., and evaluated by one-way analysis of variance (ANOVA) or two-way ANOVA followed by Bonferroni’s post hoc test. A P value of (6)-pentazocine (Mori et al., in press), therefore, we expected that potencies to produce antinociceptive effects of these drugs were almost same. Thus, further examination was required to explore these issues. In contrast, (1)-pentazocine did not exert any antinociceptive effects under the present conditions (data not shown). A previous report showed that (1)-pentazocine diminished systemic morphine-, U-50,488H- (a jopioid receptor agonist), or other opioid receptor agonist-induced antinociceptive effects in mice (Mei and Pasternak, 2002), however, there was no big difference in the characteristics of the antinociceptive effects between (6)-pentazocine and (–)-pentazocine. (–)-Pentazocine activates antinociceptive signaling mediated through opioid receptors on the cellular membrane, whereas (1)-pentazocine may regulate the chaperone activity of Sig-1Rs at the ER (Hayashi and Su, 2007). Thus, the time lag should be considered to understand why no difference was observed in the characteristics of the antinociceptive effects between (6)-pentazocine and (–)-pentazocine. Therefore, (1)-pentazocine was administered 15 min before treatment with (–)-pentazocine to evaluate the antinociceptive effects induced by (–)-pentazocine at 20 (early phase) and 120 (delayed phase) min (Fig. 2a). (1)-Pentazocine (3 and 10 mg/kg) significantly
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Fig. 3. Increase in the association between l-opioid receptor (lOR) and sigma-1 receptor (Sig-1R) by morphine in CHO cells. CHO cells transiently expressed Sig-1R and l-OR, and were treated with morphine (0.3 lM). At 1, 4 and 18 h after the administration of morphine, l-ORs were coimmunoprecipitated with Sig-1 Rs (a, b). **P < 0.01, vs. morphine-treated for 0-h group. Changes in the association between l-OR and Sig-1R by the administration of (1)-pentazocine (0.3 lM) and NE-100 (1 lM) with or without morphine (0.3 lM) (c). Immunoglobulin G did not associate with l-OR (d).
Fig. 2. Effects of (1)-pentazocine on (–)-pentazocine-induced antinociceptive effects in mice. The antinociceptive effects were evaluated at 20 and 120 min after the injection of (–)-pentazocine (a). Effects of pretreatment with NE-100 on the inhibition of the (– )-pentazocine-induced antinociceptive effects by (1)-pentazocine in mice. Mice were treated with NE-100 before the injection of (1)and (–)-pentazocine. The antinociceptive effects were evaluated at 20 min after the administration of (–)-pentazocine. Each point and column represents the mean with SEM of 10–14 animals. *P < 0.05 versus (–)-pentazocine-alone group. ##P < 0.01 versus (–)-pentazocine and (1)-pentazocine injection group (b).
attenuated the (–)-pentazocine (15 mg/kg)-induced antinociceptive effects at 20 min (F3, 49 5 6.261, P < 0.01), but not 120 min. To further confirm the involvement of Sig-1R in the suppressive effects of Sig-1R agonist against the antinociceptive effects of (–)-pentazocine, we used the selective Sig-1R antagonist NE-100. NE-100 (1–5.6 mg/kg) significantly antagonized the suppressive effects of (1)-pentazocine on the antinociceptive effects of (–)-pentazocine (F3, 40 5 7.412, P < 0.01) (Fig. 2b). These results suggest that (1)-pentazocine suppressed the antinociceptive effects of (–)-pentazocine through Sig-1Rs. As mentioned above, Sig-1R acts as an ER chaperone protein or regulates signal transduction by regulating l-opioid receptor-Sig-1R interactions. Previous
co-immunoprecipitation studies have shown that Sig1R ligands regulate channel-Sig-1R interaction at the cellular membrane (Kourrich et al., 2013) and ankyrin-Sig-1R interaction at the ER (Hayashi and Su, 2001). Recently, Kim et al. (2010) demonstrated that Sig-1Rs are physically associated with l-opioid receptors, which regulate G-protein-coupled receptor signaling. Therefore, we hypothesized that Sig-1R agonist might dissociate Sig-1R-l-opioid receptor interactions at the cellular membrane. To examine whether l-opioid receptors and Sig-1Rs physically interact with each other, co-immunoprecipitation studies were performed using HA-tagged l-opioid receptor and FLAGtagged Sig-1R that were transiently overexpressed in CHO cells. There is no commercially available l-opioid receptor antibody for immunoprecipitation, and the Sig-1R antibody that we used elsewhere was not good enough for immunoprecipitation in our preliminarily study. Therefore, we decided to overexpress the FLAGor HA-tagged proteins for immunoprecipitation in CHO cells in this study. Consistent with previous results (Kim et al., 2010), l-opioid receptors were coimmunoprecipitated with Sig-1R. Interestingly, this association between l-opioid receptor and Sig-1Rs was significantly increased at 4 h after the administration Synapse
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of morphine (300 nM) (P < 0.01) (Fig. 3a,b). Here, Sig-1R as a chaperone protein should associate with l-opioid receptors at the ER. However, our preliminary data showed that l-opioid receptors levels were decreased by morphine in CHO cells overexpressing l-opioid receptors, indicating that morphine does not increase the production of l-opioid receptors at the ER. Therefore, it is likely that increase in association between l-opioid receptors and Sig-1R occurs in the cellular membrane. Unfortunately, these increased associations between l-opioid receptor and Sig-1R by morphine were not affected by 10 min pretreatment with (1)-pentazocine or NE-100 (Fig. 3c), indicating that Sig-1R ligands can not affect the Sig1R-l-opioid receptor complex. DISCUSSION The present and a previous study (Suzuki et al., 1991) have demonstrated that both (6)-pentazocine and (–)-pentazocine produce biphasic antinociceptive effects as measured by warm-plate tests at 51 C. Previously, we showed that the antinociceptive effects in the early phase induced by (6)-pentazocine are mediated by the activation of l-opioid receptors, whereas those in the delayed phase are mediated by j-opioid receptors (Suzuki et al., 1991). While there is almost no evidence available regarding how pentazocine metabolites can act as agonists for opioid receptors, (6)-pentazocine was rapidly cleared from the blood (el Maati and Way, 1971). Therefore, it is possible that metabolites of (–)-pentazocine may act as an agonist for j-opioid receptors to produce antinociceptive effects. In the present study, the Sig-1R agonist (1)pentazocine attenuated the early phase, but not the delayed phase, of the antinociceptive effects of (–)pentazocine. Similarly, (1)-pentazocine can attenuate morphine-induced antinociceptive effects in mice (Chien and Pasternak, 1994). These results suggest that (1)-pentazocine may suppress signal transduction mediated through the activation of l-opioid receptors to produce antinociceptive effects. Recent studies have shown that Sig-1R agonistic activity could act as a nociceptive stimulus under several conditions (e.g., NMDA-mediated nociception (Yoon et al., 2010), capsaicin-induced nociception (Entrena et al., 2009) and diabetes-induced allodynia (Ohsawa et al., 2010)). The mechanism(s) that underlies the Sig-1R agonist-induced suppressive effect on the antinociceptive effects of l-opioid receptor agonist or nociceptive stimuli is still unclear. Kim et al. (2010) showed that Sig-1R antagonist enhanced GTPgS binding induced by l-opioid receptor agonist in mouse brain membrane and neuroblastoma cells, which naturally express opioid receptors (Kim et al., 2010). Furthermore, an increase in GTPgS binding induced by m-opioid receptor agonist was potently enhanced by the knock-down of Sig-Rs by siRNA in neuroblastoma cells. The present as well as previous Synapse
results (Kim et al., 2010) showed that Sig-1R could physically associate with l-opioid receptor. Furthermore, the present study also suggested that the association between Sig-1R and m-opioid receptor was increased in the presence of morphine. These results suggest that Sig-1Rs negatively regulate m-opioid receptor signal transduction by their association with m-opioid receptors and G-proteins. Most Sig-1R resides inside the ER, and the chaperone activity of Sig-1R has been shown to be regulated by protein-protein interaction between Sig-1R and BiP at the ER to combat against ER stress (Hayashi and Su, 2007). However, under certain conditions, Sig-1R could translocate to the cellular membrane (Kourrich et al., 2013; Su et al., 2010; Su et al., 2009; Yao et al., 2010). Therefore, one possible explanation for how Sig1R agonist suppresses the antinociceptive effects of (– )-pentazocine or exerts nociceptive effects is that Sig1R agonists can inhibit Sig-1R-m-opioid receptor association, which in turn produces nociceptive effects by inhibiting the m-opioid receptor to regulate signal transduction. However, (1)-pentazocine did not affect the association between overexpressed Sig-1R and lopioid receptor, suggesting that Sig-1R agonist may not affect the physical association between Sig-1Rs and l-opioid receptors. Thus, Sig-1R agonists can suppress the antinociceptive effects of (–)-pentazocine by some mechanism other than reduction of the Sig-1R-mopioid receptor association (e.g., Sig-1R agonist may suppress the modulation of m-opioid receptor signaling by regulating a certain step, such as by regulating the Ca21 channel at the ER, NMDA-receptor, or Kv1.2 channels on the cellular membrane). In contrast, the activation of G-protein by the l-opioid receptor agonist [d-Ala2, N-Me-Phe4, Gly5-ol] enkephalin (DAMGO) was potently enhanced by knocking-down of Sig-1R or Sig1R antagonist (Kim et al., 2010). Therefore, it is possible that Sig-1R itself may regulate l-opioid receptor signal transduction by regulating G-protein or other related proteins, but not l-opioid receptor. Therefore, further studies will be required to elucidate these issues. In conclusion, (1)-pentazocine attenuated the antinociceptive effects of (–)-pentazocine by regulating m-, but not j-opioid receptor-mediated functions. Furthermore, the innate antinociceptive action of (6)-pentazocine could be marginally reduced by the effects of (1)-pentazocine under our present conditions. However, (6)-pentazocine is clinically used under different conditions and in different ways, and therefore the innate antinociceptive action of (–)-pentazocine could be reduced by the anti-antinociceptive action of (1)-pentazocine. ACKNOWLEDGMENTS There is no conflict of interest with any financial organization regarding the material discussed in the manuscript.
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REFERENCES Chien CC, Pasternak GW. 1994. Selective antagonism of opioid analgesia by a sigma system. J Pharmacol Exp Ther 271:1583– 1590. Cormaci G, Mori T, Hayashi T, Su TP. 2007. Protein kinase A activation down-regulates, whereas extracellular signal-regulated kinase activation up-regulates sigma-1 receptors in B-104 cells: Implication for neuroplasticity. J Pharmacol Exp Ther 320:202–210. el-Mazati AM, Way EL. 1971 The biologic disposition of pentazocine in the rat. J Pharmacol Exp Ther 177 332–341. Entrena JM, Cobos EJ, Nieto FR, Cendan CM, Baeyens JM, Del Pozo E. 2009. Antagonism by haloperidol and its metabolites of mechanical hypersensitivity induced by intraplantar capsaicin in mice: Role of sigma-1 receptors. Psychopharmacology (Berl) 205: 21–33. Fontanilla D, Johannessen M, Hajipour AR, Cozzi NV, Jackson MB, Ruoho AE. 2009. The hallucinogen N,N-dimethyltryptamine (DMT) is an endogenous sigma-1 receptor regulator. Science 323: 934–937. Gutstein HB, Akil H. 2001. Opioid analgesics. In: Hardman JG, Limbird LE, Gilman AG, editors, Goodman & Gilman’s The Pharmacological Basis of Therapeutics, Tenth Edition. New York: McGraw-Hill. p 569–619. Hayashi T, Su TP. 2001. Regulating ankyrin dynamics: Roles of sigma-1 receptors. Proc Natl Acad Sci U S A. 98:491–496. Epub 2001 Jan 9. Hayashi T, Su TP. 2007. Sigma-1 receptor chaperones at the ERmitochondrion interface regulate Ca(21) signaling and cell survival. Cell 131:596–610. Kim FJ, Kovalyshyn I, Burgman M, Neilan C, Chien CC, Pasternak GW. 2010. Sigma 1 receptor modulation of G-protein-coupled receptor signaling: Potentiation of opioid transduction independent from receptor binding. Mol Pharmacol 77:695–703. Kourrich S, Hayashi T, Chuang JY, Tsai SY, Su TP, Bonci A. 2013. Dynamic interaction between sigma-1 receptor and Kv1.2 shapes neuronal and behavioral responses to cocaine. Cell 152:236–247. Marrazzo A, Parenti C, Scavo V, Ronsisvalle S, Scoto GM, Ronsisvalle G. 2006. In vivo evaluation of (1)-MR200 as a new selective sigma ligand modulating MOP, DOP and KOP supraspinal analgesia. Life Sci 78:2449–2453.
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Mei J, Pasternak GW. 2002. Sigma1 receptor modulation of opioid analgesia in the mouse. J Pharmacol Exp Ther 300:1070–1074. Mei J, Pasternak GW. 2007. Modulation of brainstem opiate analgesia in the rat by sigma 1 receptors: A microinjection study. J Pharmacol Exp Ther 322:1278–1285. Mori T, Itoh Y, Yoshizawa K, Ise Y, Mizuo K, Saeki T, Masukawa D, Shibasaki M, Suzuki T. Involvement of l- and d-opioid receptor function in the rewarding effect of (6)-pentazocine Addict Biol (in press). Navarro G, Moreno E, Aymerich M, Marcellino D, McCormick PJ, Mallol J, Cortes A, Casado V, Canela EI, Ortiz J, Fuxe K, Lluis C, Ferre S, Franco R. 2010. Direct involvement of sigma-1 receptors in the dopamine D1 receptor-mediated effects of cocaine. Proc Natl Acad Sci U S A 107:18676–18681. Ohsawa M, Hayashi SS, Kamei J. 2010. Effect of acute topical application of 1-pentazocine on the mechanical allodynia in diabetic mice. Eur J Pharmacol 641:49–53. Su TP, Hayashi T. 2003. Understanding the molecular mechanism of sigma-1 receptors: Towards a hypothesis that sigma-1 receptors are intracellular amplifiers for signal transduction. Curr Med Chem 10:2073–2080. Su TP, Hayashi T, Maurice T, Buch S, Ruoho AE. 2010. The sigma1 receptor chaperone as an inter-organelle signaling modulator. Trends Pharmacol Sci 31:557–566. Su TP, Hayashi T, Vaupel DB. 2009. When the endogenous hallucinogenic trace amine N,N-dimethyltryptamine meets the sigma-1 receptor. Sci Signal 2:pe12. Suzuki T, Narita M, Misawa M, Nagase H. 1991. Pentazocineinduced biphasic analgesia in mice. Life Sci 48:1827–1835. Tseng LF, Hogan QH, Wu HE. 2011. (1)-Morphine attenuates the (–)-morphine-produced tail-flick inhibition via the sigma-1 receptor in the mouse spinal cord. Life Sci 89:875–877. Yao H, Yang Y, Kim KJ, Bethel-Brown C, Gong N, Funa K, Gendelman HE, Su TP, Wang JQ, Buch S. 2010. Molecular mechanisms involving sigma receptor-mediated induction of MCP-1: Implication for increased monocyte transmigration. Blood 115: 4951–4962. Yoon SY, Roh DH, Seo HS, Kang SY, Moon JY, Song S, Beitz AJ, Lee JH. 2010. An increase in spinal dehydroepiandrosterone sulfate (DHEAS) enhances NMDA-induced pain via phosphorylation of the NR1 subunit in mice: involvement of the sigma-1 receptor. Neuropharmacology 59:460–467.
Synapse
Effects Of (1)-Pentazocine on the Antinociceptive Effects of (–)-Pentazocine in Mice TOMOHISA MORI, JUNPEI OHYA, TOSHIMASA ITOH, YUYA ISE, MASAHIRO SHIBASAKI, AND TSUTOMU SUZUKI* Department of Toxicology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, Tokyo, Japan
KEY WORDS
pentazocine; l-opioid receptor; sigma-1 receptor; antinociception
ABSTRACT Previous studies have shown that sigma-1 receptor chaperone (Sig1R) ligands can regulate pain-related behaviors, and Sig-1R itself can regulate lopioid receptor functions as well as signal transduction. Even though (6)-pentazocine has been used clinically for the treatment of pain through opioid receptors, (1)-pentazocine is known to be a selective Sig-1R agonist. To the best of our knowledge, there is no information available regarding the involvement of Sig-1R agonistic action in the antinociceptive effects of (6)-pentazocine. Therefore, the present study was designed to investigate the effects of (1)-pentazocine on the antinociceptive effects of (–)-pentazocine in mice. Both and (–)-pentazocine induced biphasic antinociceptive effects as measured by the warm-plate test. The early phase, but not the delayed phase, of the antinociceptive effects induced by (–)-pentazocine, which are mediated by the activation of l-opioid receptors, were suppressed by pretreatment with (1)-pentazocine. These results suggest that the innate antinociceptive action of (6)-pentazocine could be marginally reduced by the effects of (1)-pentazocine, but (1)-pentazocine can suppress the antinociceptive effects of (–)-pentazocine at certain time points. Synapse 69:166–171, 2015. VC 2015 Wiley Periodicals, Inc. INTRODUCTION Sigma-1 receptor (Sig-1R) was originally considered to be an opioid receptor based on its displacement of the SKF10047 binding site, and thus far it has been defined as a non-opioid binding site, unlike l-, d-, and j-opioid receptors (Su and Hayashi, 2003). Over the past few years, our understanding of Sig-1R receptors has been changing; Sig-1R is mainly localized at one of the endoplasmic reticulum (ER) domains, and is a ligand-operated ER-chaperone protein that regulates ER stress to promote the proper folding of newly synthesized or unfolded protein (Hayashi and Su, 2007). Furthermore, recent findings have demonstrated that Sig-1R can associate with G-protein coupled receptors or channels, and then regulate the signaling cascade (Cormaci et al., 2007; Kim et al., 2010; Kourrich et al., 2013; Navarro et al., 2010; Su et al., 2009). A large and growing body of evidence has demonstrated that Sig-1Rs agonists exert several pharmacological effects, such as nociceptive, hallucinogenic, neuroprotective and anti-depressant effects. However, the underlying mechanisms by which Sig-1R agonist induces pharmacological effects is still unclear. Although pentazocine has enantiomers, called (1)pentazocine and (–)-pentazocine, the racemic comÓ 2015 WILEY PERIODICALS, INC.
pound (6)-pentazocine has been used for the management of mild to moderate pain in humans. (–)Pentazocine exerts its antinociceptive effects mainly mediated by the activation of opioid receptors (Gutstein and Akil, 2001). Most j-opioid receptor agonists induce dysphoric and psychotomimetic effects, and a high dose of (6)-pentazocine also elicits dysphoric and psychotomimetic effects (Gutstein and Akil, 2001). On the other hand, (1) pentazocine is known to be a selective Sig-1R agonist. The prototypical Sig1R agonist SKF10047 induces hallucinogenic/psychotomimetic effects, and the endogenous hallucinogenic amine N,N-dimethyltryptamine was recently identified as an endogenous sigma-receptor ligand (Fontanilla et al., 2009). Thus, the (1)-pentazocine component of (6)-pentazocine may have a negative Contract grant sponsor: Ministry of Education, Culture, Sports, Science and Technology of Japan; Contract grant sponsor: JSPS KAKENHI; Contract grant number: 23590651. *Correspondence to: Tsutomu Suzuki, Department of Toxicology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan. E-mail: [email protected] Received 29 August 2014; Revised 20 November 2014; Accepted 27 November 2014 DOI: 10.1002/syn.21799 Published online 6 January 2015 in Wiley Online Library (wileyonlinelibrary. com).
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influence on the pharmacological profile of (–)pentazocine. Previous studies have shown that Sig-1R antagonist could enhance opioid receptor agonist-induced antinociceptive effects (Marrazzo et al., 2006; Mei and Pasternak, 2007). Conversely, Sig-1R agonists could have compromising effects on opioid receptor agonist-induced antinociceptive effects under certain conditions (Entrena et al., 2009; Ohsawa et al., 2010; Tseng et al., 2011). However, little or no information is available regarding how (1)-pentazocine affects the pharmacological, and especially the antinociceptive effects, of (–)-pentazocine. Therefore, the present study was designed to investigate the effects of (1)pentazocine on the antinociceptive effects of (-)-pentazocine using warm-plate tests. MATERIALS AND METHODS Animals Male ddY mice (Tokyo Laboratory Animals Science Co. Ltd, Tokyo, Japan), weighing 25–30 g, were housed in groups of 10 in a temperature-controlled (23 6 1 C) specific pathogen free room. The animals were maintained on a 12-h light/dark cycle (lights on 8:00 a.m. to 8:00 p.m.) with laboratory chow and tap water available ad libitum. This study was conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals, Hoshi University School of Pharmacy and Pharmaceutical Sciences, as adopted by the Committee on Animal Research of Hoshi University, which is accredited by the Ministry of Education, Science, Sports and Culture of Japan. Warm-plate test The antinociceptive response was evaluated by recording the latency to paw licking or tapping in the warm-plate test (51 6 0.5 C; Muromachi Kikai, Tokyo, Japan), as described previously (Suzuki et al., 1991). To prevent tissue damage, we established a 30 sec cut-off time. The test was performed after treatment with pentazocine. Each animal served as its own control, and the latency to response was measured both before and after drug administration. In the combination study, (1)-pentazocine was pretreated 15 min before the administration of (–)-pentazocine. NE-100 was also pretreated 15 min before the administration of (1)-pentazocine. Antinociception was calculated as the percentage of the maximum possible effect (% antinociception) according to the following formula: % antinociception 5 (test latency – pre-drug latency)/ (cut-off time – pre-drug latency) 3 100. Cell cultures and treatments Chinese hamster ovary (CHO) cells, which have endogenous Sig-1R, but not l-opioid receptor, were maintained in 75-cm2 flasks (Falcon; BD Biosciences
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Discovery Labware, Bedford, MA) in 10% FBScontaining Minimum Essential Medium Alpha medium at 37 C with 5% CO2 under saturated humidity. The medium was changed every 1–2 days. For drug treatments, cells seeded in 6-well plates were incubated with drugs dissolved in distilled H2O. Controls received the same amount of the vehicle. For transfection, the vector (2–3 lg per well)/Lipofectamine-2000 (6 ll per well) (Invitrogen, Carlsbad, CA) mixture was applied to CHO cells in 6-well plates. After incubation for 4–5 h, the medium was replaced. Western blotting After treatment with drugs, cells were placed on ice, rapidly washed once with ice-cold phosphate-buffered saline (PBS) and harvested in PBS with a rubber policeman. Cell suspensions were centrifuged at 800g for 10 min at 4 C. The supernatants were discarded and the pellets were dissolved with lysis buffer (10 mM Tris-HCl, pH 5 7.5, 150 mM NaCl, 0.5 % Triton X-100, supplemented with a protease inhibitor (Roche Diagnostics, Tokyo, Japan)). Cell lysates were cleared by centrifugation at 20000g at 4 C for 10 min. After deglycosylation with N-glycosidase F (Roche Diagnostics, Tokyo, Japan), cell lysates were denatured by 53 Laemmli sample buffer [250 mM Tris-HCl, pH 5 6.8, 20% glycerol, 10% sodium dodecyl sulfate (SDS)] with b-mercaptoethanol and bromophenol blue overnight at 4 C. Proteins (15–100 lg) were resolved by polyacrylamide SDS-PAGE. Gels were electroblotted onto PVDF membranes (GE Healthcare, Tokyo, Japan) in Towbeen buffer (25 mM Tris-base, 192 mM glycine). Membranes were blocked with 0.5% non-fat dry milk (Bio-Rad) in Tris-buffered saline plus 0.05% Tween20 (TBS-T) for 1h at room temperature. Membranes were incubated overnight at 4 C with an antibody in TBS-T, and probed with the secondary anti-rabbit or anti-mouse immunoglobulin G conjugated with horseradish peroxidase for 1.5 h at room temperature (1:10000 dilution). Protein bands were visualized with a SuperSignal West Femt reagent (Thermo Fisher Scientific K.K., Kanagawa, Japan) with a Fluor Chem 3 system (Laboratory and Medical Supplies, Tokyo, Japan). Anti-FLAG (Sigma-Aldrich Japan K.K., Tokyo, Japan) and anti-HA (Cell Signaling Technology, Tokyo, Japan) were purchased. Coimmunoprecipitation For Sigma-1 receptor-FLAG immunoprecipitation, cell pellets were lysed for 30 min in 150 ll of lysis buffer [10 mM Tris-HCl, pH 5 7.5, 150 mM NaCl, 0.5% Triton X-100, supplemented with a protease inhibitor (Roche Diagnostics, Tokyo, Japan)]. Cell lysates were cleared by centrifugation at 20000g at 4 C for 10 min. Fifteen micro liters of agarosecoupled anti-FLAGM2 antibody (Sigma-Aldrich Japan Synapse
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a volume of 10 ml/kg. Pentazocines were dissolved in water containing 30% lactate acid and 20% saline. Data analysis Data are expressed as the mean 6 S.E.M., and evaluated by one-way analysis of variance (ANOVA) or two-way ANOVA followed by Bonferroni’s post hoc test. A P value of (6)-pentazocine (Mori et al., in press), therefore, we expected that potencies to produce antinociceptive effects of these drugs were almost same. Thus, further examination was required to explore these issues. In contrast, (1)-pentazocine did not exert any antinociceptive effects under the present conditions (data not shown). A previous report showed that (1)-pentazocine diminished systemic morphine-, U-50,488H- (a jopioid receptor agonist), or other opioid receptor agonist-induced antinociceptive effects in mice (Mei and Pasternak, 2002), however, there was no big difference in the characteristics of the antinociceptive effects between (6)-pentazocine and (–)-pentazocine. (–)-Pentazocine activates antinociceptive signaling mediated through opioid receptors on the cellular membrane, whereas (1)-pentazocine may regulate the chaperone activity of Sig-1Rs at the ER (Hayashi and Su, 2007). Thus, the time lag should be considered to understand why no difference was observed in the characteristics of the antinociceptive effects between (6)-pentazocine and (–)-pentazocine. Therefore, (1)-pentazocine was administered 15 min before treatment with (–)-pentazocine to evaluate the antinociceptive effects induced by (–)-pentazocine at 20 (early phase) and 120 (delayed phase) min (Fig. 2a). (1)-Pentazocine (3 and 10 mg/kg) significantly
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Fig. 3. Increase in the association between l-opioid receptor (lOR) and sigma-1 receptor (Sig-1R) by morphine in CHO cells. CHO cells transiently expressed Sig-1R and l-OR, and were treated with morphine (0.3 lM). At 1, 4 and 18 h after the administration of morphine, l-ORs were coimmunoprecipitated with Sig-1 Rs (a, b). **P < 0.01, vs. morphine-treated for 0-h group. Changes in the association between l-OR and Sig-1R by the administration of (1)-pentazocine (0.3 lM) and NE-100 (1 lM) with or without morphine (0.3 lM) (c). Immunoglobulin G did not associate with l-OR (d).
Fig. 2. Effects of (1)-pentazocine on (–)-pentazocine-induced antinociceptive effects in mice. The antinociceptive effects were evaluated at 20 and 120 min after the injection of (–)-pentazocine (a). Effects of pretreatment with NE-100 on the inhibition of the (– )-pentazocine-induced antinociceptive effects by (1)-pentazocine in mice. Mice were treated with NE-100 before the injection of (1)and (–)-pentazocine. The antinociceptive effects were evaluated at 20 min after the administration of (–)-pentazocine. Each point and column represents the mean with SEM of 10–14 animals. *P < 0.05 versus (–)-pentazocine-alone group. ##P < 0.01 versus (–)-pentazocine and (1)-pentazocine injection group (b).
attenuated the (–)-pentazocine (15 mg/kg)-induced antinociceptive effects at 20 min (F3, 49 5 6.261, P < 0.01), but not 120 min. To further confirm the involvement of Sig-1R in the suppressive effects of Sig-1R agonist against the antinociceptive effects of (–)-pentazocine, we used the selective Sig-1R antagonist NE-100. NE-100 (1–5.6 mg/kg) significantly antagonized the suppressive effects of (1)-pentazocine on the antinociceptive effects of (–)-pentazocine (F3, 40 5 7.412, P < 0.01) (Fig. 2b). These results suggest that (1)-pentazocine suppressed the antinociceptive effects of (–)-pentazocine through Sig-1Rs. As mentioned above, Sig-1R acts as an ER chaperone protein or regulates signal transduction by regulating l-opioid receptor-Sig-1R interactions. Previous
co-immunoprecipitation studies have shown that Sig1R ligands regulate channel-Sig-1R interaction at the cellular membrane (Kourrich et al., 2013) and ankyrin-Sig-1R interaction at the ER (Hayashi and Su, 2001). Recently, Kim et al. (2010) demonstrated that Sig-1Rs are physically associated with l-opioid receptors, which regulate G-protein-coupled receptor signaling. Therefore, we hypothesized that Sig-1R agonist might dissociate Sig-1R-l-opioid receptor interactions at the cellular membrane. To examine whether l-opioid receptors and Sig-1Rs physically interact with each other, co-immunoprecipitation studies were performed using HA-tagged l-opioid receptor and FLAGtagged Sig-1R that were transiently overexpressed in CHO cells. There is no commercially available l-opioid receptor antibody for immunoprecipitation, and the Sig-1R antibody that we used elsewhere was not good enough for immunoprecipitation in our preliminarily study. Therefore, we decided to overexpress the FLAGor HA-tagged proteins for immunoprecipitation in CHO cells in this study. Consistent with previous results (Kim et al., 2010), l-opioid receptors were coimmunoprecipitated with Sig-1R. Interestingly, this association between l-opioid receptor and Sig-1Rs was significantly increased at 4 h after the administration Synapse
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of morphine (300 nM) (P < 0.01) (Fig. 3a,b). Here, Sig-1R as a chaperone protein should associate with l-opioid receptors at the ER. However, our preliminary data showed that l-opioid receptors levels were decreased by morphine in CHO cells overexpressing l-opioid receptors, indicating that morphine does not increase the production of l-opioid receptors at the ER. Therefore, it is likely that increase in association between l-opioid receptors and Sig-1R occurs in the cellular membrane. Unfortunately, these increased associations between l-opioid receptor and Sig-1R by morphine were not affected by 10 min pretreatment with (1)-pentazocine or NE-100 (Fig. 3c), indicating that Sig-1R ligands can not affect the Sig1R-l-opioid receptor complex. DISCUSSION The present and a previous study (Suzuki et al., 1991) have demonstrated that both (6)-pentazocine and (–)-pentazocine produce biphasic antinociceptive effects as measured by warm-plate tests at 51 C. Previously, we showed that the antinociceptive effects in the early phase induced by (6)-pentazocine are mediated by the activation of l-opioid receptors, whereas those in the delayed phase are mediated by j-opioid receptors (Suzuki et al., 1991). While there is almost no evidence available regarding how pentazocine metabolites can act as agonists for opioid receptors, (6)-pentazocine was rapidly cleared from the blood (el Maati and Way, 1971). Therefore, it is possible that metabolites of (–)-pentazocine may act as an agonist for j-opioid receptors to produce antinociceptive effects. In the present study, the Sig-1R agonist (1)pentazocine attenuated the early phase, but not the delayed phase, of the antinociceptive effects of (–)pentazocine. Similarly, (1)-pentazocine can attenuate morphine-induced antinociceptive effects in mice (Chien and Pasternak, 1994). These results suggest that (1)-pentazocine may suppress signal transduction mediated through the activation of l-opioid receptors to produce antinociceptive effects. Recent studies have shown that Sig-1R agonistic activity could act as a nociceptive stimulus under several conditions (e.g., NMDA-mediated nociception (Yoon et al., 2010), capsaicin-induced nociception (Entrena et al., 2009) and diabetes-induced allodynia (Ohsawa et al., 2010)). The mechanism(s) that underlies the Sig-1R agonist-induced suppressive effect on the antinociceptive effects of l-opioid receptor agonist or nociceptive stimuli is still unclear. Kim et al. (2010) showed that Sig-1R antagonist enhanced GTPgS binding induced by l-opioid receptor agonist in mouse brain membrane and neuroblastoma cells, which naturally express opioid receptors (Kim et al., 2010). Furthermore, an increase in GTPgS binding induced by m-opioid receptor agonist was potently enhanced by the knock-down of Sig-Rs by siRNA in neuroblastoma cells. The present as well as previous Synapse
results (Kim et al., 2010) showed that Sig-1R could physically associate with l-opioid receptor. Furthermore, the present study also suggested that the association between Sig-1R and m-opioid receptor was increased in the presence of morphine. These results suggest that Sig-1Rs negatively regulate m-opioid receptor signal transduction by their association with m-opioid receptors and G-proteins. Most Sig-1R resides inside the ER, and the chaperone activity of Sig-1R has been shown to be regulated by protein-protein interaction between Sig-1R and BiP at the ER to combat against ER stress (Hayashi and Su, 2007). However, under certain conditions, Sig-1R could translocate to the cellular membrane (Kourrich et al., 2013; Su et al., 2010; Su et al., 2009; Yao et al., 2010). Therefore, one possible explanation for how Sig1R agonist suppresses the antinociceptive effects of (– )-pentazocine or exerts nociceptive effects is that Sig1R agonists can inhibit Sig-1R-m-opioid receptor association, which in turn produces nociceptive effects by inhibiting the m-opioid receptor to regulate signal transduction. However, (1)-pentazocine did not affect the association between overexpressed Sig-1R and lopioid receptor, suggesting that Sig-1R agonist may not affect the physical association between Sig-1Rs and l-opioid receptors. Thus, Sig-1R agonists can suppress the antinociceptive effects of (–)-pentazocine by some mechanism other than reduction of the Sig-1R-mopioid receptor association (e.g., Sig-1R agonist may suppress the modulation of m-opioid receptor signaling by regulating a certain step, such as by regulating the Ca21 channel at the ER, NMDA-receptor, or Kv1.2 channels on the cellular membrane). In contrast, the activation of G-protein by the l-opioid receptor agonist [d-Ala2, N-Me-Phe4, Gly5-ol] enkephalin (DAMGO) was potently enhanced by knocking-down of Sig-1R or Sig1R antagonist (Kim et al., 2010). Therefore, it is possible that Sig-1R itself may regulate l-opioid receptor signal transduction by regulating G-protein or other related proteins, but not l-opioid receptor. Therefore, further studies will be required to elucidate these issues. In conclusion, (1)-pentazocine attenuated the antinociceptive effects of (–)-pentazocine by regulating m-, but not j-opioid receptor-mediated functions. Furthermore, the innate antinociceptive action of (6)-pentazocine could be marginally reduced by the effects of (1)-pentazocine under our present conditions. However, (6)-pentazocine is clinically used under different conditions and in different ways, and therefore the innate antinociceptive action of (–)-pentazocine could be reduced by the anti-antinociceptive action of (1)-pentazocine. ACKNOWLEDGMENTS There is no conflict of interest with any financial organization regarding the material discussed in the manuscript.
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