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J. Biol. Chem., Vol. 282, Issue 41, 29803-29811, October 12, 2007

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Long-Acting Opioid Antagonists Disrupt Receptor Signaling And Produce Noncompetitive Effects By Activating C-Jun N-Terminal Kinase^*

From the Department of Pharmacology, University of Washington, Seattle, Washington 98195

Norbinaltorphimine (NorBNI), guanidinonaltrindole, and atrans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl) piperidine (JDTic) are selective 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 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.

Received for publication, July 6, 2007 , and in revised form, August 14, 2007.

^* Funding was provided by National Institute on Drug Abuse, United States Public Health Service Grants R37-DA11672 and KO5-DA20570 (to C. C.) and F32-DA20430 (to M. R. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Traditional Chinese Medical University, 9th Bldg. 50 Shi Dong Rd., Guiyang, Guizhou, postal code 550002, China.

² Present addresses: AstraZeneca B1541, 1800 Concord Pike, P. O. Box 15437, Wilmington, DE 19820-5437.

³ To whom correspondence should be addressed: Dept. of Pharmacology, University of Washington, Box 357280, 1959 Pacific Ave. N. E., Seattle, WA 98195-7280. Tel.: 206-543-4266; Fax: 206-685-3822; E-mail: cchavkin{at}u.washington.edu.

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