μ-Opioid receptor down-regulation and tolerance are not equally dependent upon G-protein signaling

Abstract

In the present study, the contribution of pertussis toxin (PTX)-sensitive G_i/o-proteins to opioid tolerance and μ-opioid receptor down-regulation in the mouse were examined. Mice were injected once intracerebroventricularly and intrathecally with PTX (0.1 μg/site). Controls were treated with saline. On the 10th day following PTX treatment, continuous subcutaneous infusion of etorphine (150 or 200 μg/kg/day) or morphine (40 mg/kg/day+25 mg slow-release pellet) was begun. Control mice were implanted with inert placebo pellets. Pumps and pellets were removed 3 days later, and mice were tested for morphine analgesia or μ-opioid receptor density was determined in the whole brain, spinal cord, and midbrain. Both infusion doses of etorphine produced significant tolerance (ED₅₀ shift=≈4–6-fold) and down-regulation of μ-opioid receptors (≈20–35%). Morphine treatment also produced significant tolerance (ED₅₀ shift=≈5–8-fold), but no μ-opioid receptor down-regulation. PTX dramatically reduced the acute potency of morphine and blocked the further development of tolerance by both etorphine and morphine treatments. However, PTX had no effect on etorphine-induced μ-opioid receptor down-regulation in brain, cord, or midbrain. These results suggest that PTX-sensitive G-proteins have a minimal role in agonist-induced μ-opioid receptor density regulation in vivo, but are critical in mediating acute and chronic functional effects of opioids such as analgesia and tolerance.

Introduction

Activation of guanine–nucleotide binding proteins (G-proteins) is the first step in the cascade of receptor-mediated effects of G-protein-coupled receptors (GPCR), including the opioid receptors (e.g., Sanchez-Blazquez et al., 1995, Rossi et al., 1995, Raffa et al., 1994). Numerous studies indicate that opioid receptors are coupled to pertussis toxin (PTX)-sensitive G_i/o-proteins Shah et al., 1997, Goode and Raffa, 1997, Hoehn et al., 1988. PTX catalyzes the ADP ribosylation of a cysteine side chain on the α-subunit of G_i/o-proteins (Resine, 1990) and inactivates it. Antisense targeting studies confirm the importance of G-proteins in opioid receptor signaling and have shown that G_iα-subunits play a dominant role in opioid coupling to intracellular events (Standifer et al., 1996). However, the role that G-proteins play in chronic opioid effects such as the regulation of opioid receptor density and tolerance is less understood.

Down-regulation of opioid receptors is readily observed following chronic exposure to high-intrinsic-efficacy opioid agonists (e.g., etorphine), but not following low-intrinsic-efficacy agonists (e.g., morphine) Shen et al., 2000, Zaki et al., 2000, Whistler et al., 1999, Yabaluri and Medzihradsky, 1997, Duttaroy and Yoburn, 1995. While, it is clear that down-regulation is not required for opioid tolerance Whistler et al., 1999, Duttaroy and Yoburn, 1995, evidence suggests that it contributes to the magnitude of opioid tolerance Shen et al., 2000, Stafford et al., 2001. As such, it is important to understand the events that mediate opioid receptor down-regulation and its contribution to chronic opioid effects such as tolerance.

Several investigators have examined the role that PTX-sensitive G-proteins play in opioid receptor internalization and down-regulation in cell culture. In some cases, agonist-induced receptor regulation is independent of G-protein function Law et al., 1985, Kato et al., 1998, Remmers et al., 1998; while other reports indicate that receptor regulation may be partially blocked by PTX Zaki et al., 2000, Yabaluri and Medzihradsky, 1997, Chakrabarti et al., 1997 and that sensitivity differs for μ- and δ-opioid receptors (e.g., Chakrabarti et al., 1997). These cell culture results make it difficult to predict if PTX-sensitive G-proteins are critical in opioid receptor regulation in the intact animal. On the other hand, the functional effects of opioid agonists in cell culture and in vivo (e.g., inhibition of adenylyl cyclase, analgesia) are uniformly inhibited by interference with G-protein function (e.g., Remmers et al., 1998, Shen et al., 1998).

In the present study, we investigated the role of PTX-sensitive G-proteins in opioid effects in the intact animal. Agonist-induced changes in μ-opioid receptor density, opioid agonist potency, and tolerance following PTX treatment were examined. We show that G-protein function does not impact on μ-opioid receptor down-regulation, but plays a significant role in opioid tolerance.