Analgesic tolerance of opioid agonists in mutant mu-opioid receptors expressed in sensory neurons following intrathecal plasmid gene delivery

Molecular Pain

Li-Yen Mae Huang²  Yanping Gu²  Fei Ma¹  Guangwen Li² 

[1] Department of Physiology, University of Kentucky, 800 Rose Street, Lexington, KY 40536-0298, USA;Department of Neuroscience and Cell Biology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1069, USA

关键词: Plasmid DNA injection;    Nociception;    Dorsal root ganglion;    T394A mutation;    Opioid receptors;    Opioid tolerance;   

Others  :  862071
DOI  :  10.1186/1744-8069-9-63

 received in 2013-03-01, accepted in 2013-11-22,  发布年份 2013

【 摘 要 】

Background

Phosphorylation sites in the C-terminus of mu-opioid receptors (MORs) are known to play critical roles in the receptor functions. Our understanding of their participation in opioid analgesia is mostly based on studies of opioid effects on mutant receptors expressed in in vitro preparations, including cell lines, isolated neurons and brain slices. The behavioral consequences of the mutation have not been fully explored due to the complexity in studies of mutant receptors in vivo. To facilitate the determination of the contribution of phosphorylation sites in MOR to opioid-induced analgesic behaviors, we expressed mutant and wild-type human MORs (hMORs) in sensory dorsal root ganglion (DRG) neurons, a major site for nociceptive (pain) signaling and determined morphine- and the full MOR agonist, DAMGO,-induced effects on heat-induced hyperalgesic behaviors and potassium current (I_K) desensitization in these rats.

Findings

A mutant hMOR DNA with the putative phosphorylation threonine site at position 394 replaced by an alanine (T394A), i.e., hMOR-T, or a plasmid containing wild type hMOR (as a positive control) was intrathecally delivered. The plasmid containing GFP or saline was used as the negative control. To limit the expression of exogenous DNA to neurons of DRGs, a neuron-specific promoter was included in the plasmid. Following a plasmid injection, hMOR-T or hMOR receptors were expressed in small and medium DRG neurons. Compared with saline or GFP rats, the analgesic potency of morphine was increased to a similar extent in hMOR-T and hMOR rats. Morphine induced minimum I_K desensitization in both rat groups. In contrast, DAMGO increased analgesic potency and elicited I_K desensitization to a significantly less extent in hMOR-T than in hMOR rats. The development and extent of acute and chronic tolerance induced by repeated morphine or DAMGO applications were not altered by the T394A mutation.

Conclusions

These results indicate that phosphorylation of T394 plays a critical role in determining the potency of DAMGO-induced analgesia and I_K desensitization, but has limited effect on morphine-induced responses. On the other hand, the mutation contributes minimally to both DAMGO- and morphine-induced behavioral tolerance. Furthermore, the study shows that plasmid gene delivery of mutant receptors to DRG neurons is a useful strategy to explore nociceptive behavioral consequences of the mutation.

【 授权许可】

   
2013 Li et al.; licensee BioMed Central Ltd.

【 图 表 】

【 参考文献 】

• [1]Dang VC, Christie MJ: Mechanisms of rapid opioid receptor desensitization, resensitization and tolerance in brain neurons. Br J Pharmacol 2012, 165:1704-1716.
• [2]Williams JT, Ingram SL, Henderson G, Chavkin C, von Zastrow M, Schulz S, Koch T, Evans CJ, Christie MJ: Regulation of mu-opioid receptors: desensitization, phosphorylation, internalization, and tolerance. Pharmacol Rev 2013, 65:223-254.
• [3]Dang VC, Williams JT: Chronic morphine treatment reduces recovery from opioid desensitization. J Neurosci 2004, 24:7699-7706.
• [4]Deng HB, Yu Y, Pak Y, O’Dowd BF, George SR, Surratt CK, Uhl GR, Wang JB: Role for the C-terminus in agonist-induced mu opioid receptor phosphorylation and desensitization. Biochemistry 2000, 39:5492-5499.
• [5]Wang H, Guang W, Barbier E, Shapiro P, Wang JB: Mu opioid receptor mutant, T394A, abolishes opioid-mediated adenylyl cyclase superactivation. Neuroreport 2007, 18:1969-1973.
• [6]Wolf R, Koch T, Schulz S, Klutzny M, Schroder H, Raulf E, Buhling F, Hollt V: Replacement of threonine 394 by alanine facilitates internalization and resensitization of the rat mu opioid receptor. Mol Pharmacol 1999, 55:263-268.
• [7]Schulz S, Mayer D, Pfeiffer M, Stumm R, Koch T, Hollt V: Morphine induces terminal micro-opioid receptor desensitization by sustained phosphorylation of serine-375. EMBO J 2004, 23:3282-3289.
• [8]Grecksch G, Just S, Pierstorff C, Imhof AK, Gluck L, Doll C, Lupp A, Becker A, Koch T, Stumm R, Hollt V, Schulz S: Analgesic tolerance to high-efficacy agonists but not to morphine is diminished in phosphorylation-deficient S375A mu-opioid receptor knock-in mice. J Neurosci 2011, 31:13890-13896.
• [9]Hylden JL, Wilcox GL: Intrathecal morphine in mice: a new technique. Eur J Pharmacol 1980, 67:313-316.
• [10]Duttaroy A, Kirtman R, Farrell F, Phillips M, Philippe J, Monderson T, Yoburn BC: The effect of cumulative dosing on the analgesic potency of morphine in mice. Pharmacol Biochem Behav 1997, 58:67-71.
• [11]Gu Y, Xu Y, Li GW, Huang LY: Remote nerve injection of Mu opioid receptor adeno-associated viral vector increases antinociception of intrathecal morphine. J Pain 2005, 6:447-454.
• [12]Birdsong WT, Arttamangkul S, Clark MJ, Cheng K, Rice KC, Traynor JR, Williams JT: Increased agonist affinity at the mu-opioid receptor induced by prolonged agonist exposure. J Neurosci 2013, 33:4118-4127.
• [13]Hughes TS, Langer SJ, Johnson KW, Chavez RA, Watkins LR, Milligan ED, Leinwand LA: Intrathecal injection of naked plasmid DNA provides long-term expression of secreted proteins. Mol Ther 2009, 17:88-94.
• [14]Milligan ED, Sloane EM, Langer SJ, Hughes TS, Jekich BM, Frank MG, Mahoney JH, Levkoff LH, Maier SF, Cruz PE, Flotte TR, Johnson KW, Mahoney MM, Chavez RA, Leinwand LA, Watkins LR: Repeated intrathecal injections of plasmid DNA encoding interleukin-10 produce prolonged reversal of neuropathic pain. Pain 2006, 126:294-308.
• [15]Chen YJ, Oldfield S, Butcher AJ, Tobin AB, Saxena K, Gurevich VV, Benovic JL, Henderson G, Kelly E: Identification of phosphorylation sites in the COOH-terminal tail of the mu-opioid receptor. J Neurochem 2013, 124:189-199.
• [16]Doll C, Konietzko J, Poll F, Koch T, Hollt V, Schulz S: Agonist-selective patterns of micro-opioid receptor phosphorylation revealed by phosphosite-specific antibodies. Br J Pharmacol 2011, 164:298-307.
• [17]Qiu Y, Law PY, Loh HH: Mu-opioid receptor desensitization: role of receptor phosphorylation, internalization, and representation. J Biol Chem 2003, 278:36733-36739.
• [18]Pak Y, O’Dowd BF, George SR: Agonist-induced desensitization of the mu opioid receptor is determined by threonine 394 preceded by acidic amino acids in the COOH-terminal tail. J Biol Chem 1997, 272:24961-24965.
• [19]Walwyn WM, Wei W, Xie CW, Chiu K, Kieffer BL, Evans CJ, Maidment NT: Mu opioid receptor-effector coupling and trafficking in dorsal root ganglia neurons. Neuroscience 2006, 142:493-503.
• [20]Bailey CP, Oldfield S, Llorente J, Caunt CJ, Teschemacher AG, Roberts L, McArdle CA, Smith FL, Dewey WL, Kelly E, Henderson G: Involvement of PKC alpha and G-protein-coupled receptor kinase 2 in agonist-selective desensitization of mu-opioid receptors in mature brain neurons. Br J Pharmacol 2009, 158:157-164.
• [21]Doll C, Poll F, Peuker K, Loktev A, Gluck L, Schulz S: Deciphering micro-opioid receptor phosphorylation and dephosphorylation in HEK293 cells. Br J Pharmacol 2012, 167:1259-1270.
• [22]Kelly E: The subtleties of micro-opioid receptor phosphorylation. Br J Pharmacol 2011, 164:294-297.
• [23]Zheng H, Chu J, Zhang Y, Loh HH, Law PY: Modulating micro-opioid receptor phosphorylation switches agonist-dependent signaling as reflected in PKCepsilon activation and dendritic spine stability. J Biol Chem 2011, 286:12724-12733.
• [24]Levitt ES, Williams JT: Morphine desensitization and cellular tolerance are distinguished in rat locus ceruleus neurons. Mol Pharmacol 2012, 82:983-992.
• [25]Dang VC, Chieng B, Azriel Y, Christie MJ: Cellular morphine tolerance produced by betaarrestin-2-dependent impairment of mu-opioid receptor resensitization. J Neurosci 2011, 31:7122-7130.
• [26]Quillinan N, Lau EK, Virk M, von Zastrow M, Williams JT: Recovery from mu-opioid receptor desensitization after chronic treatment with morphine and methadone. J Neurosci 2011, 31:4434-4443.
• [27]Xu Y, Gu Y, Xu GY, Wu P, Li GW, Huang LY: Adeno-associated viral transfer of opioid receptor gene to primary sensory neurons: a strategy to increase opioid antinociception. Proc Natl Acad Sci U S A 2003, 100:6204-6209.
• [28]Huang LY, Neher E: Ca2+-dependent exocytosis in the somata of dorsal root ganglion neurons. Neuron 1996, 17:135-145.
• [29]Wang C, Gu Y, Li GW, Huang LY: A critical role of the cAMP sensor Epac in switching protein kinase signalling in prostaglandin E2-induced potentiation of P2X3 receptor currents in inflamed rats. J Physiol 2007, 584:191-203.