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 ϩ one 25-mg implant pellet). Controls were implanted with a placebo pellet. On the 7th day after implantation mice were tested for i. determined. Both etorphine and morphine produced significant tolerance (ED 50 shift ϭ 7.6-and 7.3-fold for morphine and etorphine, respectively). Etorphine decreased spinal OR density by Ϸ30%, whereas morphine did not change OR density. Etorphine increased (Ϸ70%) 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

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Although opioid receptors are G-protein coupled, the role that specific G-protein subunits play in the development of opioid tolerance and the regulation of opioid receptor number is not well understood. In the present study, we used a G((i)alpha2) antisense oligodeoxynucleotide (ODN) to examine the contribution of G((i)alpha2) proteins to mu-opioid tolerance and receptor downregulation in the mouse. Mice were injected intracerebroventricularly (ICV) and into the spinal intrathecal space (IT) for 4-5 consecutive days (30 microg/site/day), with an antisense ODN or a mismatch ODN directed at mRNA for the G((i)alpha2) subunit of G-proteins. Controls were treated with dH(2)O. On the second day of ODN treatment continuous subcutaneous (SC) infusion of etorphine (200 microg/kg/day) or morphine (40 mg/kg/day + 25 mg pellet) was begun. Control mice were implanted with inert placebo pellets. Three days later, pumps and pellets were removed and mice were tested for morphine analgesia or mu-opioid receptor density was determined in whole brain. Etorphine produced significant tolerance (ED(50) shift = approximately 11-fold) and downregulation of mu-opioid receptors (approximately 25%). Morphine treatment produced significant tolerance (ED(50) shift approximately 9-fold), but no mu-opioid receptor downregulation. Antisense treatment reduced G((i)alpha2) protein levels in striatum and spinal cord by approximately 25%. G((i)alpha2) antisense reduced the acute potency of morphine. G((i)alpha2) antisense blocked the development of tolerance to morphine treatment and reduced the development of tolerance to etorphine treatment. Antisense did not have any effect on etorphine-induced mu-opioid receptor downregulation. In another experiment, 7-day treatment with morphine or etorphine similarly increased G((i)alpha2) mRNA and protein abundance in spinal cord. Overall, these results support an important role for G((i)alpha2)-protein in the acute effects of opioids and opioid tolerance. However, G((i)alpha2) is not required for agonist-induced mu-opioid receptor density regulation in vivo.

Chronic opioid antagonist treatment produces functional supersensitivity and mu-opioid receptor (muOR) upregulation. Studies suggest a role for G-protein receptor kinases (GRKs) and dynamin (DYN), but not signaling proteins (e.g., G(ialpha2)), in regulation of muOR density following opioid treatment. Therefore, this study examined muOR density, agonist potency, and the abundance and gene expression of GRK-2, DYN-2, and G(ialpha2) in mouse spinal cord after opioid antagonist treatment. Mice were implanted with a 15 mg naltrexone (NTX) or placebo pellet and 8 days later pellets were removed. At 24 and 192 h following NTX treatment, mice were tested for spinal DAMGO analgesia. Other mice were sacrificed at 0 or 192 h following NTX treatment and G(ialpha2), GRK-2, and DYN-2 protein and mRNA levels determined. [(3)H] DAMGO binding studies were also conducted. Immediately following NTX treatment (0 h), muOR density was increased (+ approximately 135%), while 192 h following NTX treatment muOR density was unchanged. NTX increased DAMGO analgesic potency (3.1-fold) 24 h following NTX treatment, while there was no effect at 192 h. NTX decreased protein and mRNA abundance of GRK-2 (-32%; -48%) and DYN-2 (-25%; -29%) in spinal cord at 0 h. At 192 h following 8-day NTX treatment, GRK-2 protein and mRNA were at control levels, while DYN-2 protein remained decreased (-31%) even though DYN-2 mRNA had returned to control levels. G(ialpha2) was unaffected by NTX treatment. These data suggest that opioid antagonist-induced mu-receptor upregulation is mediated by changes in abundance and gene expression of proteins implicated in receptor trafficking, which may decrease constitutive receptor cycling.