Chronic opioid antagonist treatment dose-dependently regulates mu-opioid receptors and trafficking proteins in vivo.

Chronic opioid antagonist treatment increases the density of mu-opioid receptors (muOR) in many model systems. In previous studies, naltrexone treatment produced an increase in muOR density accompanied by decreases in GRK-2 and DYN-2 protein abundance. To examine the relationship between changes in receptor density and proteins involved in receptor trafficking, the dose-dependent effect of chronic naloxone infusion was determined. Dose-dependent antagonism of morphine analgesia was also examined. Mice were infused with naloxone (0.1, 1.0, 5.0 mg/kg/day sc) for 7 days via osmotic pump. Controls were treated with placebo pellets. On the 7th day, morphine dose-response studies were determined using the tail flick. Other mice were sacrificed at the end of the treatment and spinal cords were collected for determination of muOR density and GRK-2 and DYN-2 protein abundance. Naloxone infusion dose-dependently increased spinal muOR density with no change in affinity. The increases in mu-receptor density were proportional to dose-dependent decreases in GRK-2 and DYN-2 protein levels. Furthermore, naloxone dose-dependently antagonized morphine. These data suggest that opioid antagonist-induced muOR up-regulation in mouse spinal cord is associated with regulation of proteins involved in receptor trafficking and support suggestions that opioid antagonist-induced receptor up-regulation is due to reduced constitutive internalization of opioid receptors.

Chronic opioid antagonist treatment selectively regulates trafficking and signaling proteins in mouse spinal cord.

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.

Opioid agonists differentially regulate mu-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 mu-opioid receptor (muOR) down-regulation. In vitro studies implicate dynamin-2 (DYN-2) and G-protein 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 microg/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(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) analgesia. In other mice, spinal cord was removed for [(3)H]DAMGO binding studies or GRK-2 and DYN-2 protein and mRNA abundance were determined. Both etorphine and morphine produced significant tolerance (ED(50) shift = 7.6- and 7.3-fold for morphine and etorphine, respectively). Etorphine decreased spinal muOR density by approximately 30%, whereas morphine did not change muOR density. Etorphine increased ( approximately 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.