Abstract

Chronic morphine treatment has been shown to produce constitutive activation of μ-opioid receptors, and this transition might contribute to the development of tolerance and dependence. The apparent ability of chronic morphine to increase the spontaneous, agonist-independent activation of μ-opioid receptors may be unique, due to its distinct partial agonist properties of possessing a relatively high intrinsic activity coupled with a poor ability to produce desensitization and down-regulation. Therefore, the present study tested the hypothesis that prolonged exposure to morphine would produce greater constitutive activity of μ-opioid receptors than exposure to the full agonist [d-Ala²,N-MePhe⁴,Gly-ol⁵]enkephalin (DAMGO). GH₃ cells expressing μ-opioid receptors were exposed to chronic morphine, DAMGO, or no opioid under conditions determined to produce maximal desensitization, down-regulation, and cAMP rebound. After chronic treatment, the μ-opioid antagonists naloxone and β-chlornaltrexamine (β-CNA) were evaluated in two assays predictive of inverse agonist activity. Both antagonists produced a concentration-dependent inhibition of [³⁵S]GTPγS binding only in membranes prepared from cells chronically exposed to opioids. This effect was reversed by the neutral μ-opioid antagonist CTAP. Additionally, conditions known to uncouple G protein-coupled receptors from G proteins produced a leftward shift in the competition curve of β-CNA for [³H]DAMGO binding only in membranes prepared from chronically treated cells. In contrast, these conditions produced no shift in the competition curve by the neutral antagonist CTAP in cells exposed to chronic DAMGO. Therefore, prolonged exposure of GH₃MOR cells to opioids produced constitutive activation of μ-opioid receptors. Surprisingly, chronic treatment with the more efficacious agonist DAMGO produced greater increases in both measures of inverse agonist activity than did morphine. These observations may lend novel insight into the mechanisms of opioid tolerance and dependence.