Opioid regulation of the mouse delta-opioid receptor expressed in human embryonic kidney 293 cells

Abstract

Opioid analgesics are used extensively in the management of pain. Although the clinically effective opioids bind with high affinity to the mu-opioid receptor, studies have suggested that the delta-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 delta-receptor agonist regulation, the cloned mouse delta 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 delta-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 delta receptors. [D-Pen2,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 delta 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 delta receptor from adenylyl cyclase. In contrast to the desensitization induced by peptide-selective delta agonists, pretreatment with the delta-selective nonpeptide agonist 7-spiroindanyloxymorphone and morphine sensitized the opioid inhibition of forskolin-stimulated cAMP accumulation. This differential regulation of the delta 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 delta-selective agonists to inhibit cAMP accumulation while increasing the potency of morphine to reduce cAMP accumulation. In particular, a lack of desensitization of the delta receptor by morphine may contribute to our understanding of the molecular basis of development of morphine-induced tolerance and dependence.