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

Abstract

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.

μ-Opioid Receptor Up-Regulation and Functional Supersensitivity Are Independent of Antagonist Efficacy

“…3D.). These data with naloxone and naltrexone are consistent with earlier studies (e.g., Patel et al, 2003;Rajashekara et al, 2003), whereas up-regulation following 6␤-naltrexol is a novel finding. Naloxone and 6␤-naltrexol were equipotent in producing Ϸ2-fold increase in morphine potency (Fig.…”

“…4, left). Increases in the functional potency of opioid agonists following antagonist treatment have been reported often (e.g., Patel et al, 2003;Rajashekara et al, 2003). Overall, these findings demonstrate that inverse agonists and a neutral antagonist are equieffective in up-regulating -opioid receptors and producing functional supersensitivity.…”

“…An extension of the importance of efficacy in regulating -opioid receptors is the effect of opioid antagonists on receptor density. Numerous studies have reported that treatment with antagonists, which by definition are neutral or negative in efficacy, will substantially increase the density of -opioid receptors and produce a shift to the left of the agonist dose-response function (functional supersensitivity, e.g., Patel et al, 2003). Taken together, these data indicate that regulation of -opioid receptors occurs over a continuum with increases in density observed for antagonists, little change in density for lower efficacy agonists, and internalization and down-regulation reported for higher efficacy agonists.…”

“…Both opioid agonists and antagonists have been shown to regulate -opioid receptor density (Chakrabarti et al, 1997;Zaki et al, 2000;Rajashekara et al, 2003), and these changes can have an impact on the potency of opioid agonists (e.g., Stafford et al, 2001;Patel et al, 2003). The efficacy of an opioid agonist has been proposed to play an important role in -opioid receptor regulation (Patel et al, 2002;Pawar et al, 2007).…”

“…Conversely, low-efficacy agonists (e.g., morphine, oxycodone) are less likely to produce either internalization or down-regulation (Keith et al, 1998;Pawar et al, 2007;however, see Haberstock-Debic et al, 2005). Chronic treatment with opioid antagonists (e.g., naltrexone, naloxone) has been shown to up-regulate -, ␦-, and -opioid receptors Lesscher et al, 2003;Patel et al, 2003) and increase the potency of opioid agonists (functional supersensitivity) (Yoburn and Inturrisi, 1988;Yoburn et al, 1989Yoburn et al, , 1995.…”