Long-Acting κ Opioid Antagonists Disrupt Receptor Signaling And Produce Noncompetitive Effects By Activating C-Jun N-Terminal Kinase
2007; Elsevier BV; Volume: 282; Issue: 41 Linguagem: Inglês
10.1074/jbc.m705540200
ISSN1083-351X
AutoresMichael R. Bruchas, Tao Yang, Selena Schreiber, Mia C. DeFino, Steven C. Kwan, Shuang Li, Charles Chavkin,
Tópico(s)Pharmacological Receptor Mechanisms and Effects
ResumoNorbinaltorphimine (NorBNI), guanidinonaltrindole, and atrans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl) piperidine (JDTic) are selective κ opioid receptor (KOR) antagonists having very long durations of action in vivo despite binding non-covalently in vitro and having only moderately high affinities. Consistent with this, we found that antagonist treatment significantly reduced the subsequent analgesic response of mice to the KOR agonist U50,488 in the tail-withdrawal assay for 14–21 days. Receptor protection assays were designed to distinguish between possible explanations for this anomalous effect, and we found that mice pretreated with the readily reversible opioid antagonists naloxone or buprenorphine before norBNI responded strongly in the tail-flick analgesia assay to a subsequent challenge with U50,488 1 week later. Protection by a rapidly cleared reagent indicates that norBNI did not persist at the site of action. In vitro binding of [3H]U69,593 to KOR showed that Kd and Bmax values were not significantly affected by prior in vivo norBNI exposure, indicating that the agonist binding site was intact. Consistent with the concept that the long-lasting effects might be caused by a functional disruption of KOR signaling, both norBNI and JDTic were found to stimulate c-Jun N-terminal kinase (JNK) phosphorylation in HEK293 cells expressing KOR-GFP but not in untransfected cells. Similarly, norBNI increased phospho-JNK in both the striatum and spinal cord in wild type mice but not in KOR knock-out mice. Pretreatment of mice with the JNK inhibitor SP600125 before norBNI attenuated the long acting antagonism. Together, these results suggest that the long duration KOR antagonists disrupt KOR signaling by activating JNK. Norbinaltorphimine (NorBNI), guanidinonaltrindole, and atrans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl) piperidine (JDTic) are selective κ opioid receptor (KOR) antagonists having very long durations of action in vivo despite binding non-covalently in vitro and having only moderately high affinities. Consistent with this, we found that antagonist treatment significantly reduced the subsequent analgesic response of mice to the KOR agonist U50,488 in the tail-withdrawal assay for 14–21 days. Receptor protection assays were designed to distinguish between possible explanations for this anomalous effect, and we found that mice pretreated with the readily reversible opioid antagonists naloxone or buprenorphine before norBNI responded strongly in the tail-flick analgesia assay to a subsequent challenge with U50,488 1 week later. Protection by a rapidly cleared reagent indicates that norBNI did not persist at the site of action. In vitro binding of [3H]U69,593 to KOR showed that Kd and Bmax values were not significantly affected by prior in vivo norBNI exposure, indicating that the agonist binding site was intact. Consistent with the concept that the long-lasting effects might be caused by a functional disruption of KOR signaling, both norBNI and JDTic were found to stimulate c-Jun N-terminal kinase (JNK) phosphorylation in HEK293 cells expressing KOR-GFP but not in untransfected cells. Similarly, norBNI increased phospho-JNK in both the striatum and spinal cord in wild type mice but not in KOR knock-out mice. Pretreatment of mice with the JNK inhibitor SP600125 before norBNI attenuated the long acting antagonism. Together, these results suggest that the long duration KOR antagonists disrupt KOR signaling by activating JNK. Portoghese et al. (1Portoghese P.S. Lipkowski A.W. Takemori A.E. J. Med. Chem. 1987; 30: 238-239Crossref PubMed Scopus (138) Google Scholar, 2Portoghese P.S. Lipkowski A.W. Takemori A.E. Life Sci. 1987; 40: 1287-1292Crossref PubMed Scopus (507) Google Scholar) first reported the synthesis of the selective KOR 4The abbreviations used are: ANOVA, analysis of variance; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; GNTI, guanidinonaltrindole; GPCR, G-protein-coupled receptor; JDTic, atrans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl) piperidine; KOR, κ opioid receptor; MOR, μ opioid peptide receptor; JNK, c-Jun N-terminal Kinase; MAPK, mitogen-activated protein kinase; norBNI, norbinaltorphimine HCl; TBS, Tris-buffered saline; TBST, Tris-buffered saline with Tween 20; GTPγS, guanosine 5′-3-O-(thio)triphosphate. antagonist Norbinaltorphimine (norBNI) two decades ago, and this ligand has been the most commonly used KOR antagonist since. NorBNI has a greater than 100-fold selectivity for KOR over the μ or δ opioid receptors (MOR and DOR, respectively) (3Metcalf M.D. Coop A. AAPS J. 2005; 7: E704-E722Crossref PubMed Scopus (91) Google Scholar). KOR is a G-protein-coupled receptor (GPCR) that is widely expressed throughout the nervous system and is activated by endogenous opioid peptide agonists derived from prodynorphin (4Chavkin C. James J.F. Goldstein A. Science. 1982; 215: 413-415Crossref PubMed Scopus (1045) Google Scholar, 5Dhawan B.N. Cesselin R. Raghubir T. Reisine P.B. Portoghese P.S. Hamon M. Pharmcol. Rev. 1996; 48: 568-586Google Scholar). Several reports have shown that agonist occupation of the KOR leads to the pertussis toxin-sensitive inhibition of adenylate cyclase, increase in potassium conductance, decrease in calcium conductance, and mobilization of intracellular calcium (6Piros E.T. Hales T.G. Evans C.J. Neurochem. Res. 1996; 21: 1277-1285Crossref PubMed Scopus (38) Google Scholar). Recently, KOR activation has also been shown to stimulate the mitogen-activated protein kinase pathways (MAPK), including extracellular signal-regulated kinase (ERK1/2), p38, and c-Jun N-terminal Kinase (JNK) (7Belcheva M.M. 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GNTI and JDTic have similar long-lasting effects and produce antagonism for at least 10–14 days (12Negus S.S. Mello N.K. Linsenmayer D.C. Jones R.M. Portoghese P.S. Psychopharmacology (Berl.). 2002; 163: 412-419Crossref PubMed Scopus (48) Google Scholar, 13Carroll, I., Thomas, J. B., Dyskstra, L. A., Granger, A. L., Allen, R. M., Howard, J. L., Pollard, G. T., Aceto, M. D., and Harris, L. S. (2004) 501, 111–119Google Scholar). These findings are surprising because these antagonists do not covalently bind to KOR (20Smith C.B. Medzihradsky F. Hollingsworth P.J. DeCosta B. Rice K.C. Woods J.H. Prog. Clin. Biol. Res. 1990; 328: 65-68PubMed Google Scholar). The basis for this long duration of action is not clear. One explanation is that these drugs become physically trapped in the lipid membrane and do not clear easily from the nervous system. A second possibility is that these drugs are biotransformed in vivo to long-lasting metabolites that covalently bind to the receptor. An alternative hypothesis is that NorBNI, GNTI, and JDTic produce their long-lasting effects by acutely uncoupling the KOR signaling complex such that agonists can no longer activate the receptor to stimulate G-protein signaling. To distinguish these mechanisms, we first compared the duration of actions in mice for norBNI, GNTI, and JDTic. Building on these findings, we used receptor protection experiments and looked at both the functional and binding properties of KOR ligands. If transient occupancy of KOR by a readily reversible ligand could protect against receptor inactivation, the long-lasting antagonist must also produce its effects by transiently occupying the same binding site rather than by forming a drug depot in the brain. Using this strategy, we found that the readily reversible opioid antagonists naloxone and buprenorphine were able to protect KOR signaling. We further found that the long-lasting antagonists activate JNK in a KOR-dependent manner, and we found that that blockade of JNK activation significantly attenuated the long-lasting antagonism. Understanding how κ antagonists produce long-lasting effects has important implications for the ultimate utility of these agents as therapeutic tools. Recent studies have suggested that the antagonists might have antidepressant activity and also be useful in preventing relapse of drug abuse (21McLaughlin J.P. Marton-Popovici M. Chavkin C. J. Neurosci. 2003; 23: 5674-5683Crossref PubMed Google Scholar, 22Mague S.D. Pliakas A.M. Todtenkopf M.S. Tomasiewicz H.C. Zhang Y. Stevens Jr., W.C. Jones R.M. Portoghese P.S. Carlezon Jr., W.A. J. Pharmacol. Exp. Ther. 2003; 305: 323-330Crossref PubMed Scopus (409) Google Scholar, 23Beardsley P.M. Howard J.L. Shelton K.L. Carroll F.I. Psychopharmacology (Berl). 2005; 183: 118-126Crossref PubMed Scopus (233) Google Scholar). In addition, understanding how JNK activation by these drugs disrupts KOR signaling would provide new insight to opioid and GPCR signal transduction events. Chemicals—(–)U50,488, norBNI, and GNTI were obtained from Tocris (Ellisville, MO). Buprenorphine was obtained from the National Institute on Drug Abuse Drug Program (Bethesda, MD), and naloxone was from Sigma. JDTic was provided by Dr. F. I. Carroll (Research Triangle Institute, NC). All other drugs were purchased from Calbiochem. Drugs were dissolved in water or saline (for in vivo experiments) unless otherwise indicated. Animals and Housing—Male C57Bl/6 mice (Charles River Laboratories, Wilmington, MA) weighing 20–30 g (8–12 weeks old) were used in these experiments. Mice were maintained in a specific pathogen-free housing unit in the core animal facility at the University of Washington. Housing rooms were illuminated on a 12-h light-dark cycle with lights on at 7 a.m. Food pellets were available ad libitum. All animal procedures were approved by the institutional Animal Care and Use Committee in accordance with NIH guidelines. Breeding and Genotyping of MOR and KOR Knock-out Mice—Homozygous μ opioid receptor (MOR) and KOR knock-out (–/–) mice were prepared by homologous recombination as described (24Schuller A.G. King M.A. Zhang J. Bolan E. Pan Y.X. Morgan D.J. Chang A. Czick M.E. Unterwald E.M. Pasternak G.W. Pintar J.E. Nat. Neurosci. 1999; 2: 151-156Crossref PubMed Scopus (276) Google Scholar, 25Clarke S. Czyzyk T. Ansonoff M. Nitsche J.F. Hsu M.S. Nilsson L. Larsson K. Borsodi A. Toth G. Hill R. Kitchen I. Pintar J.E. Eur. J. Neurosci. 2002; 16: 1705-1712Crossref PubMed Scopus (27) Google Scholar) and provided for this study. Animals were backcrossed for >10 generations with C57Bl/6 mice, and heterozygote breeding pairs were used to make homozygote MOR–/– mice and paired littermate controls for this study. Mice were genotyped using DNA extracted from tail samples as described previously (21McLaughlin J.P. Marton-Popovici M. Chavkin C. J. Neurosci. 2003; 23: 5674-5683Crossref PubMed Google Scholar). Cell Culture—HEK293 cells were grown as previously described (26McLaughlin J.P. Xu M. Mackie K. Chavkin C. J. Biol. Chem. 2003; 278: 34631-34640Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) in Dulbecco's modified Eagle's medium/nutrient mixture F-12 with l-glutamine and 15 mm HEPES (Invitrogen) with 10% fetal bovine serum, 50 units/ml penicillin, and 50 μg/ml streptomycin at 37 °C and 5% CO2. HEK293 cells transfected with green fluorescent protein tagged KOR-GFP or MOR-GFP were maintained in the above media with an additional 200 μg/ml G418 to maintain selective pressure. Untransfected HEK293 cells for control experiments were grown in the absence of G418. Immunoblotting—KOR-GFP, MOR-GFP, and untransfected HEK293 cells were cultured as described above. Cells were serum-starved in Dulbecco's modified Eagle's medium/F-12 for 18 h before to drug treatment. Cells were treated with U50,488 (10 μm), norBNI (1 μm), anisomycin (100 μm), naloxone (1 μm), GNTI (100 nm), or vehicle control for the appropriate time periods and then immediately lysed in 50 mm Tris-HCl, 300 mm NaCl, 1 mm EDTA, 1 mm Na3VO4, 1 mm NaF, 10% glycerol, 1:100 phosphatase inhibitor mixture set 1 (Calbiochem), and 1:100 protease inhibitor mixture set 1 (Calbiochem). Lysates were sonicated (20 s, 4 °C) and centrifuged (15,000 × g, 15 min, 4 °C), and the supernatant was stored at –20 °C. For spinal cord and striatal samples, mice were injected intraperitoneally with drug as indicated, tissue was dissected (60 min after final injection), and tissue was homogenized/lysed using a 2-ml Dounce homogenizer in buffer (as above). Total protein concentration was determined using Pierce bicinchronic assay with bovine serum albumin standards before loading 40 μg onto non-denaturing 10% bisacrylamide precast gels (Invitrogen) and running at 140 V for 1.5–2 h. Blots were transferred to nitrocellulose (Whatman, Middlesex, UK) for 1.5–2 h at 30 V. The nitrocellulose was then washed with TBS (5 min), blocked with 5% milk/TBST (60 min), washed with TBST (3 × 5 min), and incubated overnight at 4 °C in phospho-stress-activated protein kinase/JNK (Thr-183/Tyr-185) rabbit antibody or phospho-ERK1/2 (Thr-202/Tyr-204) rabbit antibody diluted 1:1000 in 5% bovine serum albumin/TBST (Cell Signaling, Beverly, MA). After overnight incubation, the blots were washed with TBST (3 × 10 min) and incubated for 60 min at room temperature in anti-rabbit IRDye800 diluted 1:10,000 in a 1:1 mixture of 5% milk/TBST and Li-Cor blocking buffer (Li-Cor Biosciences, Lincoln, NE). The blots were washed with TBST (3 × 5 min) and TBS (5 min) and then scanned on the Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE). The blots were re-probed with rabbit anti-β-actin diluted 1:2500 in 5% milk/TBST (2 h, room temperature) and secondary antibody (as above) to confirm equal protein loading. In Vivo Antinociceptive Testing—The response latency for the mouse to withdraw its tail after being immersed in 52 ± 1°C water was taken as the end point. A cut-off time of 15 s was used to prevent heat-related tissue damage. Radioligand Binding—Whole mouse brains with the cerebellum removed were homogenized in ice-cold 50 mm Tris buffer, pH 7.5, followed by centrifugation at 26,000 × g for 30 min at 4 °C. Three brains were pooled for each independent replicate with the pellet collected as the membrane fraction. Homogenization and centrifugation were repeated twice more to wash the membrane fractions and remove residual ligand present in the preparation. After the final centrifugation step, excess buffer was removed, and the membranes were stored at –80 °C until use. Membranes were resuspended in ice-cold Tris buffer. Protein concentrations were determined by BCA assay. Samples were incubated for 90 min at room temperature with the KOR ligand [3H]U69,543. Nonspecific binding was determined in the presence of 10 μm U50,488. GF/B glass fiber filters (Brandel) were preincubated for 90 min at room temperature with Tris buffer, 0.3% polyethyleneimine. After 90 min of incubation at room temperature, samples were placed on ice and collected with the filters with a Brandel 24 well harvester. Filters were washed 3× with cold Tris buffer and counted in 5 ml of Ecoscint scintillation fluid. For saturation binding, [3H]U69,543 was tested at concentrations ranging from 0.156 to 20 nm. Radioligand concentrations were confirmed by scintillation count of free ligand. Data Analysis—Immunoblots were scanned using the Odyssey Infrared Imaging System (Li-Cor Bioscience, Lincoln, NE). Band intensity of both phospho-p54 and phospho-p46 (JNK1 and -2) was measured together using the Odyssey software, which subtracts background and calculates band density in pixels. Data were normalized to a percentage of control sample band intensity (basal, 100%) and plotted using GraphPad (GraphPad Prism 4.0, San Diego, CA) software. Concentrationresponse curves and saturation binding isotherms were fit using non-linear regression (Prism 4.0), and Scatchard plots were fit using linear regression analysis. Statistical significance was taken as p < 0.05 or p < 0.01 as determined by the Student's t test or ANOVA followed by a Bonferroni post hoc test where appropriate. NorBNI, GNTI, and JDTic Produce Long-lasting Antagonism for 14–21 Days—Treatment of mice with a single dose of norBNI (10 mg/kg, intraperitoneal) resulted in significant blockade of U50,488 (KOR agonist)-mediated analgesic responses 24 h through 21 days after initial injection as measured by tail withdrawal assay (Fig. 1, A and B). However, 28 days after injection, residual norBNI effects were absent, with no difference between norBNI and saline-injected groups evident at this time point. This slow rate of recovery of analgesic activity of U50,488 is the same as after treatment of mice with the receptor alkylating drug β-chlornaltrexamine (27McLaughlin J.P. Myers L.C. Zarek P.E. Caron M.G. Leftkowitz R.J. Czyzyk T.A. Pintar J.E. Chavkin C. J. Biol. Chem. 2004; 279: 1810-1818Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), and the results suggest that recovery of response requires new receptor synthesis. Further characterization of other KOR antagonists showed similar long durations of action in the analgesic assay. Single injection of GNTI (10 mg/kg) resulted in sustained and significant KOR antagonism that persisted for 21 days (Fig. 1B). JDTic (10 mg/kg intraperitoneally) also showed robust KOR antagonism for 14 days, and its effect reversed only at day 21 (Fig. 1B). Naloxone, a non-selective, competitive opioid receptor antagonist, produced transient KOR antagonism and did not block U50,488 analgesia on day 2 or during the duration of the experiment (Fig. 1B). For comparison, a single injection of the agonist U50,488 (10 mg/kg intraperitoneally) was also found not to produce long-lasting effects, demonstrating that opioid tolerance did not underlie the long-lasting antagonism by norBNI, JDTic, and GNTI. These results are consistent with prior reports and demonstrate that norBNI, GNTI, and JDTic all have long durations of antagonist activity in mice.FIGURE 1NorBNI, JDTic, and GNTI produce long-lasting antagonism in mice.A, mice were given a single injection of norBNI (10 mg/kg intraperitoneally) or saline, and tail withdrawal latencies were measured as described under “Experimental Procedures” at the specified time points 30 min after an injection of U50,488 (15 mg/kg intraperitoneally). NorBNI significantly antagonized U50,488-induced analgesic responses through day 21 after initial injection. Data are the mean tail “flick” latencies expressed as ± S.E. n = 8–12 where each n is a separate animal. *, significantly different fromU50,488 + saline group, p < 0.05, using one-way ANOVA followed by a Bonferroni post hoc test. B, mice were given a single injection of norBNI (10 mg/kg intraperitoneally), GNTI (10 mg/kg intraperitoneally), JDTic (10 mg/kg intraperitoneally), naloxone (30 mg/kg intraperitoneally), or U50,488 (15 mg/kg intraperitoneally), and then the tail withdrawal latencies were measured at the designated time points 30 min after injection of U50,488 (15 mg/kg intraperitoneally). NorBNI and GNTI had similar time courses for KOR antagonism. JDTic produced significant KOR antagonism for 14 days. Naloxone and U50,488 did not produce long-lasting effects on KOR-mediated analgesic responses. Data are the mean ± S.E. of tail flick latencies. n = 8, where each n is a separate animal. *, significantly different from saline control, p < 0.05, using one-way ANOVA followed by a Bonferroni post hoc test.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Protection of KOR with Competitive Antagonists Prevents the NorBNI Long Duration of Action—To assess whether the long duration of action of norBNI was due to slow clearance of the drug, we used a receptor protection strategy. Mice were pretreated with naloxone (30 mg/kg intraperitoneally) (non-selective competitive opioid receptor antagonist) to block available KOR binding sites before injection of norBNI. If norBNI remained in the brain, then once naloxone cleared the system, residual norBNI would again have access to KOR and cause blockade. Surprisingly, in this experiment naloxone given 15 min before norBNI (10 mg/kg intraperitoneally) significantly blocked the reduction in U50,488 response measured on day 8 after norBNI injection (Fig. 2A). To further test this result we next used buprenorphine, which is a mixed acting opioid ligand with partial agonist activity at MOR and competitive antagonist activity at KOR (28Sadee W. Rosenbaum J.S. Herz A. J. Pharmacol. Exp. Ther. 1982; 223: 157-162PubMed Google Scholar, 29Young A.M. Stephens K.R. Hein D.W. Woods J.H. J. Pharmacol. Exp. Ther. 1984; 229: 118-126PubMed Google Scholar). To eliminate the potential confounding effects of partial agonist activity at MOR, we assessed the protective effects of buprenorphine in MOR knock-out mice (MOR–/–). Injection of mice with buprenorphine (3 mg/kg intraperitoneally) 60 min before injection of norBNI (10 mg/kg) significantly protected KOR from norBNI antagonism (day 8) (Fig. 2B). Mice pretreated with buprenorphine before norBNI responded to the U50,488 challenge with the same increase in tail-flick latency as mice not pretreated with norBNI or pretreated with buprenorphine alone. Buprenorphine alone did not produce analgesia in MOR–/– mice and did not produce sustained KOR antagonism measured on day 8. Together these results suggest that the long duration of action of norBNI was not due to the pharmacokinetic properties of norBNI but, instead, by norBNI acting at the receptor to produce changes in KOR functioning. NorBNI Does Not Alter KOR Binding Characteristics—Single injections of norBNI followed by saturation binding experiments 1 week later were performed to assess whether norBNI exposure affected KOR receptor agonist affinity or KOR receptor density. Mice were injected with norBNI (10 mg/kg intraperitoneally), and brain membranes were prepared on day 8 for radioligand binding. Using the selective agonist [3H]U69,593, we found no significant difference in the total receptor density (Bmax) or the affinity for κ agonist (Kd) between norBNI-treated (Bmax = 21 ± 2 fmol/mg of protein, Kd = 1.65 ± 0.4 nm) and saline-treated (Bmax = 21 ± 1 fmol/mg of protein, Kd = 1.0 ± 0.2 nm) mouse brain membranes taken 7 days after injection of norBNI (Fig. 3A and Table 1). These data are consistent with previous reports demonstrating that norBNI does not form a covalent bond to the receptor and does not decrease apparent receptor number (20Smith C.B. Medzihradsky F. Hollingsworth P.J. DeCosta B. Rice K.C. Woods J.H. Prog. Clin. Biol. Res. 1990; 328: 65-68PubMed Google Scholar, 30Takemori A.E. Begona Ho Y.B. Neaseth J.S. Portoghese P.S. J. Pharmacol. Exp. Ther. 1988; 246: 255-258PubMed Google Scholar).TABLE 1Affinity values, total binding site densities, and pharmacological data for KOR ligands in mouse brain tissue and HEK293 cellsGroup[3H]U69,593pJNKKdBmaxEC50IAnmfmol/mgnmSaline1.00 ± 0.2021 ± 1NorBNI1.65 ± 0.4021 ± 2158 ± 180.84Buprenorphine + saline0.73 ± 0.1619 ± 1Buprenorphine + NorBNI2.50 ± 0.7823 ± 3U50,4881240 ± 1801.00Dynorphin B87 ± 130.61JDTic5 ± 30.57BuprenorphineNRNR Open table in a new tab The effect of receptor protection on membranes from norBNI-treated mice was also measured. In this experiment mice were pretreated with buprenorphine (3 mg/kg intraperitoneally) 1 h before injection of norBNI (10 mg/kg intraperitoneally), and membranes were prepared on day 8 after these initial injections. There was also no significant change in either the Bmax or Kd of [3H]U69,593 (Fig. 3B and Table 1), further supporting the conclusion that the long duration of action of norBNI cannot be attributed to changes in receptor density or affinity. NorBNI Activates c-Jun N-terminal Kinase in a KOR-dependent Manner—Previous studies have shown that norBNI acts as an antagonist for KOR-mediated signal transduction (6Piros E.T. Hales T.G. Evans C.J. Neurochem. Res. 1996; 21: 1277-1285Crossref PubMed Scopus (38) Google Scholar, 8Bohn L.M. Belcheva M.M. Coscia C. J. Neurochem. 2000; 74: 564-573Crossref PubMed Scopus (79) Google Scholar, 10Belcheva M.M. Clark A.L. Haas P.D. Serna J.S. Hahn J.W. Kiss A. Coscia C. J. Biol. Chem. 2005; 280: 27662-27669Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). For example norBNI has been shown to block the KOR-mediated inhibition of cAMP, activation of inward rectifying potassium channel, and increased ERK1/2 and p38 MAPK phosphorylation (6Piros E.T. Hales T.G. Evans C.J. Neurochem. Res. 1996; 21: 1277-1285Crossref PubMed Scopus (38) Google Scholar, 8Bohn L.M. Belcheva M.M. Coscia C. J. Neurochem. 2000; 74: 564-573Crossref PubMed Scopus (79) Google Scholar, 10Belcheva M.M. Clark A.L. Haas P.D. Serna J.S. Hahn J.W. Kiss A. Coscia C. J. Biol. Chem. 2005; 280: 27662-27669Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). While performing pilot MAPK studies in KOR-transfected cells, we were surprised to find that norBNI treatment activated JNK and hypothesized that JNK activation may be involved in its long-lasting effects. In KOR-GFP-expressing HEK293 cells, we found that treatment with U50,488 (1 h, 37 °C) caused a concentration-dependent (EC50 = 1240 ± 180 nm) increase in phospho-JNK activity. Maximal activation of JNK by U50,488 was evident at the 1-h time point, with the response returning to basal levels at 3 h (data not shown). The endogenous κ opioid peptide dynorphin (1 h, 37 °C) also increased phospho-JNK in a concentration-dependent manner (EC50 = 87 ± 13 nm) (Fig. 4, A and C, Table 1), although with a much lower efficacy than U50,488. Unexpectedly, norBNI treatment (1 h, 37 °C) also caused a concentration-dependent increase (EC50 = 158 ± 18 nm) in phospho-JNK in KOR-GFP expressing HEK293 cells (Fig. 4, B and C, Table 1). JDTic also caused a concentration-dependent (EC50 = 5 ± 3 nm) increase in phospho-JNK (Table 1) in KOR-GFP HEK293 cells. The competitive KOR antagonist, buprenorphine, however, did not elicit any JNK response in these cells, suggesting that this effect was characteristic of long acting KOR antagonists (Fig. 4, B and C, Table 1). To confirm that norBNI would produce KOR antagonism under these conditions, we measured U50,488-induced phospho-ERK1/2 activation in KOR-GFP expressing HEK293 cells. In these experiments norBNI treatment completely blocked KOR-dependent (U50,488-treated) phospho-ERK1/2 activation but in the same cells increased phospho-JNK activity (Fig. 5A). In addition, selectivity for KOR-induced phospho-JNK was confirmed by stimulating untransfected HEK293 cells with KOR ligands under the same conditions. NorBNI, JDTic, GNTI, and U50,488 (10 μm, 1 h, 37 °C) did not increase phospho-JNK in untransfected HEK293 cells (Fig. 5B). Anisomycin (100 μm, 15 min, 37 °C), the stress kinase activator, increased phospho-JNK by greater than 2-fold as expected, confirming that JNK signaling is intact in these untransfected HEK293 cells. Additionally, we stimulated MOR-GFP expressing HEK293 cells with norBNI (10 μm, 1 h, 37 °C) (Fig. 5B, inset) and found that there was no effect on phospho-JNK by possible
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