Abstract

Opioid analgesics are used extensively in the management of pain. Although the clinically effective opioids bind with high affinity to the μ-opioid receptor, studies have suggested that the δ-opioid agonists might represent more ideal analgesic agents, with fewer side effects. A limitation to opiate effectiveness is the development of tolerance, an event that has been linked to opioid receptor desensitization. To gain a better understanding of δ-receptor agonist regulation, the cloned mouse δ receptor was stably expressed in human embryonic kidney 293 cells, and the functional effects of agonist pretreatment were examined. With a 3-hr pretreatment protocol, the δ-selective agonists [d-Pen²,d-Pen⁵]enkephalin, [d-Ala²,d-Leu⁵]enkephalin, and [d-Ser²,Leu⁵]enkephalin-Thr and the nonselective opioids levorphanol, etorphine, and ethylketocyclazocine were found to desensitize δ receptors. [dPen²,d-Pen⁵]enkephalin, [d-Ser²,Leu⁵]enkephalin-Thr, [d-Ala²,d-Leu⁵]enkephalin, and etorphine treatments also caused a pronounced internalization of the epitope-tagged δ receptor, suggesting that the desensitization and internalization may be related. In contrast, levorphanol pretreatment did not internalize the receptor but still resulted in a 400-fold reduction in potency, suggesting that prolonged treatment with levorphanol only uncoupled the δ receptor from adenylyl cyclase. In contrast to the desensitization induced by peptide-selective δ agonists, pretreatment with the δ-selective nonpeptide agonist 7-spiroindanyloxymorphone and morphine sensitized the opioid inhibition of forskolin-stimulated cAMP accumulation. This differential regulation of the δ receptor may be due to variations in the ability of agonists to bind to the receptor. This hypothesis was supported by the finding that a point mutation that converted Asp128 to Asn128 (D128N) diminished the ability of δ-selective agonists to inhibit cAMP accumulation while increasing the potency of morphine to reduce cAMP accumulation. In particular, a lack of desensitization of the δ receptor by morphine may contribute to our understanding of the molecular basis of development of morphine-induced tolerance and dependence.