Opioid Agonists Differentially Regulate -Opioid Receptors and Trafficking Proteins in Vivo

Chronic opioid agonist treatment produces tolerance and in some cases opioid receptor internalization and down-regulation. Both morphine and etorphine induce tolerance; however, only etorphine produces -opioid receptor ( OR) down-regulation. In vitro studies implicate dynamin-2 (DYN-2) and Gprotein receptor kinase-2 (GRK-2) in these processes. Therefore, we examined etorphine and morphine effects on regulation of GRK-2 and DYN-2 in mouse spinal cord. Mice were treated for 7 days with etorphine (200 g/kg/day infusion) or morphine (40 mg/kg/day infusion one 25-mg implant pellet). Controls were implanted with a placebo pellet. On the 7th day after implantation mice were tested for i.t. [D-Ala,NMe-Phe,Gly-ol]-enkephalin (DAMGO) analgesia. In other mice, spinal cord was removed for [H]DAMGO binding studies or GRK-2 and DYN-2 protein and mRNA abundance were determined. Both etorphine and morphine produced significant tolerance (ED50 shift 7.6and 7.3-fold for morphine and etorphine, respectively). Etorphine decreased spinal OR density by 30%, whereas morphine did not change OR density. Etorphine increased ( 70%) DYN-2 protein abundance and decreased its mRNA (31%), whereas it had no effect on GRK-2 protein and mRNA abundance. Morphine had no effect on either DYN-2 or GRK-2 protein or mRNA abundance. These data raise the possibility that unequal receptor regulation by etorphine and morphine might be due to differential regulation of trafficking proteins. Overall, receptor down-regulation associated with chronic etorphine treatment may accelerate dynamin-related activity. Finally, the decrease in DYN-2 mRNA may be related to stabilization of DYN-2 protein abundance, which might inhibit transcription. Opioid agonists have been shown to produce tolerance and in some cases internalization and down-regulation of opioid receptors (Duttaroy and Yoburn, 1995; Keith et al., 1996; Yabaluri and Medzihradsky, 1997; Whistler et al., 1999; Shen et al., 2000; Zaki et al., 2000). The ability of opioid agonists to regulate OR has been suggested to be related to the intrinsic efficacy of the agonist. For example, chronic treatment with low intrinsic efficacy agonists (e.g., morphine) may cause blunting of receptor signaling (desensitization) without any change in surface receptor number (Yoburn et al., 1993; Burford et al., 1998; Van Bockstaele and Commons, 2001). Conversely, chronic exposure to high intrinsic efficacy opioid agonists (e.g., etorphine) can internalize and down-regulate opioid receptors, as well as produce desensitization (Duttaroy and Yoburn, 1995; Yabaluri and Medzihradsky, 1997; Whistler et al., 1999; Shen et al., 2000; Zaki et al., 2000). Internalization of OR has also been noted after acute etorphine and opioid peptide treatment (Trafton et al., 2000; Van Bockstaele and Commons, 2001). Although decreases in receptor density depend upon the agonist used, recent studies suggest that down-regulation of OR, although not required for opioid tolerance, can contribute to opioid tolerance (Stafford et al., 2001). Chronic agonist treatment has been shown to regulate G-protein-coupled receptor (GPCR) density in cell culture studies. Agonist-induced activation of G-protein receptor kinase (GRK) seems to precede receptor phosphorylation. Once the receptor has been phosphorylated, -arrestin translocates to the plasma membrane and uncouples the receptor/ G-protein complex, which may be followed by congregation of receptors in clathrin-coated pits and subsequent dynamin (DYN)-mediated internalization (Carman and Benovic, 1998; Krupnick and Benovic, 1998). Both GRK-2 and DYN-2 have been shown to play an important role in agonist-mediated internalization of OR (Zhang et al., 1998; Whistler and von Zastrow, 1998). Dominant negative mutants of GRK-2 and DYN-2 inhibit agonist-induced internalization of OR (Whistler and von Zastrow, 1998; Zhang et al., 1998; Lazari et al., 1999; Gaborik et al., 2001). Furthermore, overexpression of This work was supported by National Institutes of Health Grant DA12868. Portions of these data were presented at the annual meeting of American Society for Pharmacology and Experimental Therapeutics, April 2002. These data represent a portion of a thesis presented by M.P. to the faculty of the College of Pharmacy and Allied Health Professions, St. John’s University, in partial fulfillment of the requirements for the M.S. degree in pharmaceutical sciences. ABBREVIATIONS: OR, -opioid receptor; GPCR, G-protein-coupled receptor; GRK, G-protein receptor kinase; DYN, dynamin; DAMGO, [D-Ala,N-MePhe,Gly-ol]-enkephalin; bp, base pair(s). 0026-895X/02/6206-1464–1470$7.00 MOLECULAR PHARMACOLOGY Vol. 62, No. 6 Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics 1886/1035400 Mol Pharmacol 62:1464–1470, 2002 Printed in U.S.A.