Agonist Induced Homologous Desensitization of m-Opioid Receptors Mediated by G Protein-Coupled Receptor Kinases Is Dependent on Agonist Efficacy

Using Xenopus laevis oocytes coexpressing mammalian m-opioid receptors (MORs), b-adrenergic receptor kinase 2 (b-ARK2) [also called G protein-coupled receptor kinase (GRK3)], and b-arrestin 2 (b-arr 2), we compared the rates of b-ARK2 (GRK3)and b-arr 2-mediated homologous receptor desensitization produced by treatment with opioid agonists of different efficacies. The response to MOR activation was measured using two-electrode voltage clamp as an increase in the conductance of the coexpressed G protein-coupled inwardly rectifying potassium (heteromultimer of KIR3.1 and KIR3.4) channels. Treatment with opioids of high efficacy, either [D-Ala,NMePhe,Gly-ol]-enkephalin, fentanyl, or sufentanyl, produced a GRK3and b-arr 2-dependent reduction in response in ,20 min, whereas treatment with the partial agonist morphine produced receptor desensitization at a significantly slower rate. Because GRK3 requires activation and membrane targeting by free G protein bg subunits released after agonist-mediated activation of G proteins, a low efficacy agonist such as morphine may produce weak receptor desensitization as a consequence of poor GRK3 activation. To address this hypothesis, we substituted GRK5, a GRK that does not require activation by G protein bg. In oocytes expressing GRK5 instead of GRK3, both [D-Ala,N-MePhe,Gly-ol]enkephalin and fentanyl, but not morphine, produced desensitization of MOR-activated potassium conductance. Thus, m-opioid agonists produced significant receptor desensitization, mediated by either GRK3 or GRK5, at a rate dependent on agonist efficacy. The processes underlying the clinically observed tolerance to opioid drugs are complex and may involve learning mechanisms, compensatory changes in neuronal circuits, and desensitization of signal transduction mechanisms (Nestler and Aghajanian, 1997; Ramsay and Woods, 1997). Studies both in vivo and in vitro have documented that the response to opioids desensitize after prolonged exposure to opioids (Cox, 1978). One of the important molecular events that has been shown to be involved in the desensitization of the response to opioids is opioid receptor desensitization. The molecular basis for opioid receptor desensitization was shown to be a reduction in opioid agonist efficacy (Chavkin and Goldstein, 1982, 1984; Porreca and Burks, 1983) that manifests as a reduction in the rate of G protein activation by the agonistbound receptor complex. For example, continuous infusion of guinea pigs or rats with morphine results in an uncoupling of m-opioid receptors from associated G proteins as measured biochemically (Werling et al., 1989; Tao et al., 1993), cytochemically (Sim et al., 1996), or electrophysiologically (Christie et al., 1987). The uncoupling of MOR from G proteins is likely to result from the agonist-dependent phosphorylation of MOR mediated by a GRK (Kovoor et al. 1997). Because clinically useful opioids differ in their intrinsic efficacies, the rates of tolerance development for different opioid agonists might be expected to differ (Stevens and Yaksh ,1989a). However, the relationship between agonist efficacy and opioid tolerance is controversial. Stevens and Yaksh (1989a, 1989b) reported that tolerance to the analgesic effects of opioids measured by the hot-plate response of rats was greater for agonists with lower efficacy when continuously infused at equieffective doses. Duttaroy and Yoburn (1995) confirmed that the amount of analgesic tolerance after continuous infusion with opioids was inversely proportional to agonist efficacy but that intrinsic efficacy had no effect on the magnitude of tolerance produced by intermittent administration of opioid agonist to mice. The complexity of the pharmacological parameters controlling the in vivo situation confounds the analysis of the relationship between efficacy and tolerance because differences in receptor occupancy, drug distribution, metabolism, and This work was supported by United States Public Health Service Grant DA04123 from the National Institute on Drug Abuse. A.K. and J.P.C. contributed equally to this work. ABBREVIATIONS: MOR, m-opioid receptor; DAMGO, [D-Ala,N-MePhe,Gly-ol]enkephalin; b-ARK, b-adrenergic receptor kinase; b-arr, b-arrestin; GRK, G protein-coupled receptor kinase; HEK, human embryonic kidney; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. 0026-895X/98/040704-08$3.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 54:704–711 (1998). 704 at A PE T Jornals on M ay 7, 2017 m oharm .aspeurnals.org D ow nladed from receptor selectivity affect the measured response and subsequent changes after prolonged drug administration. Stevens and Yaksh (1989a, 1989b) explained their results by suggesting that agonists with higher efficacy had a larger fraction of spare receptors and, hence, tolerance development to the more efficacious agonists was slower. However, this assumes that agonists with different efficacies produce receptor desensitization at the same rate when they bind the same fraction of receptors. Thus, to better elucidate the molecular mechanisms underlying the relationship between efficacy and receptor desensitization, we used Xenopus laevis oocytes coexpressing the MOR and the G protein-gated inwardly rectifying potassium channel complex (KIR3.1 and KIR3.4) to reconstitute a typical neuronal opioid response (North et al., 1987). To reconstitute a homologous receptor desensitization mechanism, we also coexpressed GRKs and b-arr 2. Using this gene expression system, we previously showed that coexpression of b-ARK2 (or GRK3) and b-arr 2 synergistically produced an agonist-dependent homologous desensitization of the b2-adrenergic receptor, the d-opioid receptor, and the MOR activation of the KIR3 response (Kovoor et al., 1997). Recently, Yu and colleagues (1997) reported that opioids with high receptor efficacies were better able to stimulate MOR phosphorylation than were opioids with low efficacies. Similarly, partial agonists were previously reported to be less effective at promoting b-adrenergic receptor phosphorylation by b-ARK (i.e., GRK2) than full agonists (Benovic et al., 1988). In the current study, we asked whether m-opioid agonists with different efficacies produce GRK/b-arr 2-mediated desensitization at different rates. Materials and Methods Chemicals. DAMGO was obtained from Peninsula Laboratories (San Carlos, CA). Fentanyl, buprenorphine, and naloxone were from Research Biochemicals International (Natick, MA). Sufentanyl was a gift from Janssen Pharmaceuticals (Brussels, Belgium). All other chemicals were from Sigma Chemical (St Louis, MO). Complementary DNA clones and cRNA synthesis. Except for GRK5, the cDNA clones used and cRNA synthesis methods have been described previously (Kovoor et al., 1997). The rat MOR clone was obtained from Dr. Lei Yu (GenBank accession No, L13069). cDNA for the KIR3.1 (GIRK1) channel was obtained from Dr. Henry Lester (GenBank accession No. U01071). The KIR3.4 (GIRK4) clone provided by Dr. John Adelman (GenBank accession No. X83584) and the b-arr 2 cDNA (clone provided by Dr. Robert Lefkowitz, GenBank accession No. M91590) were first amplified by the use of Amplitaq DNA Polymerase (Perkin-Elmer Cetus, Norwalk, CT) in a standard polymerase chain reaction using oligonucleotides designed to add a T7 promoter region and a 45-base poly(A) tail. The rat GRK3 cDNA (GenBank accession No. M87855) was provided by Dr. Shaun Coughlin in the pFROGY vector. The human GRK5 clone (GenBank accession No. L15388) was obtained from Dr. Jeffrey Benovic (Kunapuli et al., 1993). Plasmid templates for all constructs were linearized before cRNA synthesis, and mMESSAGE MACHINE kits (Ambion, Austin, TX) were used to generate capped cRNA from the cDNA templates, which contained either T7, T3, or SP6 promoters to direct synthesis of sense transcripts. Oocyte culture and injection. Stage IV oocytes were prepared as described previously (Kovoor et al., 1995). cRNA was injected (50 nl/oocyte) using a Drummond automatic microinjector, and then oocytes were incubated at 18° for 3–7 days in normal oocyte saline buffer (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 5 mM HEPES, pH 7.5) solution supplemented with sodium pyruvate (2.5 mM) and gentamycin (50 mg/ml). Electrophysiology. Oocytes were clamped at 280 mV with two electrodes filled with 3 M KCl having resistances of 0.5–1.5 MV using a Geneclamp 500 amplifier and pCLAMP 6 software (Axon Instruments, Foster City, CA). Data were digitally recorded (Digidata 1200, Axon Instruments, and Intel 386PC) and filtered. Membrane current traces also were recorded using a chart recorder. To facilitate the inward potassium current flow through the KIR3 channels, normal oocyte saline buffer was modified to increase KCl concentration to 16 mM as stated in Results. All drug responses were evaluated in this high K-containing solution. In the experiments in which sufentanyl desensitization was evaluated, the high K solution in which drug responses were measured also contained 0.1% bovine serum albumin (Sigma). The concentration of NaCl was correspondingly decreased to maintain isoosmolarity. Opioid activation of KIR3 conductance was measured as described previously (Kovoor et al., 1997). Statistical analysis. The Student’s t test (with two-tailed p values) was used for comparison of the independent mean values. Doseresponse curves were fitted to a simple Emax model using the nonlinear regression analysis PCNONLIN v4.2 (SCI Software) to determine the EC50 values and 95% confidence intervals.