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, 282 (41), 29803-11

Long-acting Kappa Opioid Antagonists Disrupt Receptor Signaling and Produce Noncompetitive Effects by Activating c-Jun N-terminal Kinase

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Long-acting Kappa Opioid Antagonists Disrupt Receptor Signaling and Produce Noncompetitive Effects by Activating c-Jun N-terminal Kinase

Michael R Bruchas et al. J Biol Chem.

Abstract

Norbinaltorphimine (NorBNI), guanidinonaltrindole, and atrans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl) piperidine (JDTic) are selective kappa 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 [(3)H]U69,593 to KOR showed that K(d) 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.

Figures

FIGURE 1
FIGURE 1. NorBNI, 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.
FIGURE 2
FIGURE 2. κ opioid receptor protection by competitive antagonist reverses the sustained antagonism by norBNI
A, mice were injected with naloxone (30 mg/kg, intraperitoneal) (non-selective opioid receptor antagonist) to occlude the available KOR binding pocket to norBNI, saline, or U50,488 (15 mg/kg, intraperitoneal) before injection with norBNI (10 mg/kg, intraperitoneal), and on day 8 tail flick latencies were measured as described over a 1-h time course after U50,488 injection. Naloxone significantly protected the KOR from norBNI antagonism at the day 8 time point. *, significantly different from saline/saline group and naloxone/norBNI group, n = 4–8, p < 0.05 using one-way ANOVA, followed by a Bonferroni post hoc test. B, MOR knock-out (MOR−/−) mice were injected with buprenorphine (3 mg/kg, intraperitoneal) (KOR-selective competitive antagonist) to occlude the available KOR binding pocket to norBNI before injection with norBNI (10 mg/kg, intraperitoneal), and on day 8 tail flick latencies were measured as described over a 1-h time course. Buprenorphine significantly protected the KOR from norBNI antagonism at the day-8 time point. *, significantly different from Bup/saline (MOR−/−), Bup/NorBNI, or Bup/saline (MOR+/+). p < 0.05 using one-way ANOVA followed by Bonferroni post hoc test, n = 8, where each n is a different animal.
FIGURE 3
FIGURE 3. NorBNI does not alter KOR binding characteristics
A, mean saturation binding isotherms for specific [3H]U69,593 (a KOR-selective agonist) in membranes from mouse brain on day 8 after norBNI (10 mg/kg, intraperitoneal) or saline. Concentrations of total [3H]U69,593 ranging from 0.156 to 20 nM were incubated with membranes in the presence (nonspecific binding) and absence (total binding) of 10 μM U50,488 as described under “Experimental Procedures.” Specific binding was determined by subtracting nonspecific from total binding counts and converted into fmol/mg of protein. Non-linear regression of five individual binding curves show that [3H]U69593 bound to a homogenous receptor population and had similar affinity and receptor density in both the norBNI group (Bmax = 21 ± 1 fmol/mg of protein, Kd = 1.00 ± 0.20 nM) and the saline-treated group (Bmax = 21 ± 2 fmol/mg of protein, Kd = 1.65 ± 0.40 nM). The inset shows the mean data ± S.E. in Scatchard plots that were derived from the saturation isotherms. n = 5, where each n is a different experiment. B, mean saturation binding isotherms for specific [3H]U69,593 and measured as indicated above. Non-linear regression of five individual binding curves show that under conditions known to protect KOR from norBNI (Fig. 2), [3H]U69,593 bound to a homogenous receptor population in both the BPN (Bmax = 19 ± 1 fmol/mg of protein, Kd = 0.73 ± 0.16 nM) and BPN + NorBNI (Bmax = 23 ± 3 fmol/mg of protein, Kd = 2.50 ± 0.78 nM) groups with similar affinity and total receptor numbers. The inset shows the mean data ± S.E. in Scatchard plots that were derived from the saturation isotherms. n = 5, where each n is a different experiment.
FIGURE 4
FIGURE 4. NorBNI increases phospho-JNK in a concentration-dependent manner
A, phospho-c-Jun N-terminal kinase (pJNK) concentration response curves for the KOR agonists U50,488 and the endogenous KOR-agonist peptide dynorphin B in HEK293 cells expressing KOR-GFP. B, phospho-JNK concentration responses curves for the KOR ligands norBNI and buprenorphine in HEK293 cells expressing KOR-GFP. All ligand treatments were performed at the 1-h time point. n = 3–5, with each n taken from a separate cell culture and experiment. C, representative Western blots for phospho-JNK concentration response data in KOR-GFP-expressing HEK cells for U50,488 (top), dynorphin B (middle, Dyn B), and NorBNI (bottom).
FIGURE 5
FIGURE 5. NorBNI blocks KOR-induced ERK1/2 activation but increases phospho-JNK in KOR expressing HEK293 cells
A, mean band intensities expressed as a percentage of basal-untreated control (dashed line) ± S.E. of KOR-GFP-mediated ERK1/2 and JNK phosphorylation from HEK293 cells expressing KOR-GFP. Phospho-JNK (pJNK) but not phospho-ERK1/2 levels were significantly increased after norBNI treatment (10 μM, 1 h, 37 °C). As expected U50,488 caused a robust increase in both phospho-JNK and phospho-ERK1/2 over control. NorBNI treatment blocked U50-induced phospho-ERK1/2 activity but had no effect on U50-mediated phospho-JNK activity. * or **, significantly different from basal; p < 0.05 (*) or p < 0.01 (**)using the Student’s t test, n = 3–5, where each n is taken from an independent experiment. B, mean band intensities expressed as a percentage of basal-untreated control (dashed line) ± S.E. in untransfected HEK293 cells stimulated with the stress kinase activator anisomycin (Anis, 100 μM) or the KOR ligands NorBNI (10 μM), JDTic (10 μM), GNTI (10 μM), and U50,488 (10 μM) for 1 h at 37 °C. As predicted none of the KOR-selective ligands caused activation of JNK in cells that do not express KOR. Anisomycin treatment (50 μM, 15 min, 37 °C) increased phospho-JNK and confirmed that JNK signaling was intact in the untransfected cells. n = 3–7, where each n represents an individual experiment. *, significantly different from basal, p < 0.05 using Student’s t test. Inset, mean band intensities expressed as a percentage of basal-untreated control (dashed line) ± S.E. in MOR-GFP expressing HEK293 cells stimulated with either anisomycin or norBNI under the same conditions as above. n = 3–4, where each n represents an individual experiment.
FIGURE 6
FIGURE 6. NorBNI-induced phospho-JNK is pertussis toxin insensitive
A, mean band intensities expressed as a percentage of basal-untreated control (dashed line) ± S.E. of KOR-GFP-mediated JNK phosphorylation from HEK293 cells expressing KOR-GFP treated with pertussis toxin (200 ng/ml, 18 h, 37 °C). Phospho-JNK (pJNK) levels were significantly (*) increased 1 h after norBNI treatment and were not reversed by Gαi/o inhibition by pertussis toxin. *, significantly different from basal, p < 0.05, using Student’s t test. n = 3–4, where each n represents an individual experiment. B, representative Western blot of phospho-JNK bands in norBNI-treated KOR-GFP expressing HEK293 cells treated with and without pertussis toxin (Ptx). β-Actin Western blot below confirms equal protein loading in each lane. C, representative Western blot of phospho-ERK1/2 (pERK1/2) bands in U50,488 (U50)-treated KOR-GFP expressing HEK293 cells treated with and without pertussis toxin. β-Actin Western blot below confirms equal protein loading in each lane.
FIGURE 7
FIGURE 7. NorBNI induces JNK phosphorylation in wild type mouse spinal cord and striatum, but not in KOR−/−mice
A, mean phospho-JNK band intensities expressed as a percentage of basal-untreated control (dashed line) ± S.E. taken from spinal cord or striatal cell lysates of KOR wild type (KOR+/+) or KOR knock-out (KOR−/−) mice treated with norBNI (10 mg/kg, intraperitoneal, 1 h). NorBNI caused a significant (*) increase in phospho-JNK (pJNK) in both mouse striatum and spinal cord that was absent in samples taken from norBNI-treated KOR−/− mice. n = 5–9, where each n is taken from a separate animal; *, significantly different from basal, p < 0.05 using Student’s t test. B, representative Western blot of phospho-JNK in spinal cord cell lysate samples taken from KOR+/+ and KOR−/− norBNI or saline (SA)-treated mice. The β-actin blot below confirms equal protein loading in each lane.
FIGURE 8
FIGURE 8. Blockade of JNK reverses the long-lasting antagonism of norBNI
Wild type mice were treated with the selective JNK inhibitor SP600125 (30 mg/kg, intraperitoneal, 2 times; 24 h and 30 min before norBNI) before injection with the KOR antagonist norBNI, and tail flick analgesic responses were determined 7 days after injection. For injections, SP600125 (JNK-I) was solubilized in vehicle (V) containing 5% Me2SO4, 20% Cremophor EL, and 75% saline. U50,488 caused a significant (**) increase in the tail flick analgesic response in vehicle-treated SP600125(###) alone (JNK-I) and no vehicle (Control) groups. Inhibition of JNK before norBNI injection significantly (*) reversed the long-lasting antagonist effect on U50,488-induced analgesia. *, p < 0.05 for V/NorBNI groups versus JNK-I; **, p < 0.01 for V versus V + NorBNI groups; ##, p < 0.01 for control versus V + NorBNI groups; ###, p < 0.001 JNK-I versus V + NorBNI groups (using one-way ANOVA followed by a Bonferroni post hoc test). n = 12, where each n is a separate animal.

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