Morphine-induced mu-opioid receptor rapid desensitization is independent of receptor phosphorylation and beta-arrestins

Abstract

Receptor desensitization involving receptor phosphorylation and subsequent betaArrestin (betaArr) recruitment has been implicated in the tolerance development mediated by mu-opioid receptor (OPRM1). However, the roles of receptor phosphorylation and betaArr on morphine-induced OPRM1 desensitization remain to be demonstrated. Using OPRM1-induced intracellular Ca(2+) ([Ca(2+)](i))release to monitor receptor activation, as predicted, [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO), induced OPRM1 desensitization in a receptor phosphorylation- and betaArr-dependent manner. The DAMGO-induced OPRM1 desensitization was attenuated significantly when phosphorylation deficient OPRM1 mutants or Mouse Embryonic Fibroblast (MEF) cells from betaArr1 and 2 knockout mice were used in the studies. Specifically, DAMGO-induced desensitization was blunted in HEK293 cells expressing the OPRM1S375A mutant and was eliminated in MEF cells isolated from betaArr2 knockout mice expressing the wild type OPRM1. However, although morphine also could induce a rapid desensitization on [Ca(2+)](i) release to a greater extent than that of DAMGO and could induce the phosphorylation of Ser(375) residue, morphine-induced desensitization was not influenced by mutating the phosphorylation sites or in MEF cells lacking betaArr1 and 2. Hence, morphine could induce OPRM1 desensitization via pathway independent of betaArr, thus suggesting the in vivo tolerance development to morphine can occur in the absence of betaArr.

Fig. 1. Activation of OPRM1 induced [Ca²⁺]_i release in HEK293 cells

A, OPRM1-mediated [Ca²⁺]_i release required P₂Y receptor co-activation. Data shows the real time intracellular fluorescence change in raw fluorescence unit (RFU). HEK293 cells were cultured as described in materials and methods. Fluorescence dye to detect free [Ca²⁺]_i was added 1 hour before compound injection. After 30 second baseline reading, as indicated by the arrow, HEK293 cells expressing OPRM1 were injected with 200nM ADP (), 1µM DAMGO (□), 200nM ADP with 1µM DAMGO (), 200nM ADP, 1µM DAMGO and 30µM Naloxone () respectively. B, The concentration-response curves of DAMGO (■) and morphine (○) were determined in the presence of 200nM ADP. Total fluorescence response change induced by ADP or by ADP with OPRM1 ligands was quantified by analyzing the areas under the curves with Prism program. ADP response was then subtracted from the response in the presence of ADP and OPRM1 ligands to obtain the DAMGO and morphine responses.

Fig. 2. OPRM1 agonists induced rapid OPRM1 desensitization in HEK293 cells

A, 100nM morphine-pretreatment rapidly reduced future OPRM1 activation. In the first injection, 100nM morphine in HBSS buffer was added into pretreated groups while HBSS was added into control groups. In the second injection, 900nM morphine and 200nM ADP were added into pretreated groups, while 1µM morphine and 200nM ADP were added into control groups; thus the same final concentration of morphine and ADP in both control and pretreated groups was achieved. Then the OPRM1-mediated potentiation of ADP-induced [Ca²⁺]_i release was measured. B, Morphine and DAMGO induced OPRM1 rapid desensitization. The ability of 100nM DAMGO(○) and morphine(■) to induced desensitization of wild type OPRM1 in HEK293 cells was examined as described in Fig. 2A. Total [Ca²⁺]_i response of the second injection was quantitatively analyzed as described in Methods and legend of Figure 1. The total response in control groups of 200nM ADP and 1µM DAMGO-induced in second injection was referred as 100%; and data were expressed as the percentage of the response in the pretreated group as compare to the control group. ** denotes p< 0.01.

Fig. 3. Morphine-induced OPRM1 desensitization was resulted from loss of OPRM1 activity

A, Morphine or DAMGO pretreatment did not affect ADP-mediated response. HEK293 cells expressing wild type OPRM1 were pretreated with 1µM morphine(), DAMGO() or HBSS(▲)respectively. In second injection, various concentration of ADP together with 30µM naloxone was injected. Then the concentration-response curves of ADP were determined as described in materials and methods. B, Morphine or DAMGO pretreatment did not alter [Ca²⁺]_i store availability. HEK293 cells expressing wild type OPRM1 were cultured and seeded as described in materials and methods. After 1 hr incubation of fluorescence dye, cells were washed with Ca²⁺ free HBSS buffer. Then cells were incubated in Ca²⁺ free HBSS buffer with 1mM EGTA, and were treated with agonists. After cells were pretreated with 1µM morphine, DAMGO, 10µM ADP or HBSS respectively, 1µM thapsigargin was added. Thapsigargin-induced total fluorescence change in second injection was calculated as described in materials and methods; the data were expressed as the raw fluorescence units in bar graph. Student t-test was used to compare the data in treated groups and control group. ** denotes p< 0.01.

Fig. 4. OPRM1 agonists induced OPRM1 phosphorylation in HEK293 cells

HEK 293 cells stably expressing HA-tagged mutant or wild type OPRM1 were pretreated with 1µM morphine or 1µM DAMGO for 5 min. Receptors were immunoprecipitated and receptor phosphorylation was quantitatively analyzed as described in materials and methods. Student t-test was used to compare the data in morphine- or DAMGO-pretreated group to control group. * denotes p < 0.05; ** denotes p< 0.01.

Fig. 5. Effect of receptor phosphorylation on DAMGO- and morphine-induced OPRM1 desensitization in HEK293 cells

The ability of 100nM DAMGO and morphine to induce desensitization of wild type OPRM1(■) and phosphorylation deficient OPRM1(363/370/375)(○) was examined in HEK293 cells. Total [Ca²⁺]_i response of the second injection was quantitatively analyzed as described in figure legend of Figure 1B. The agonist-induced desensitization rate was calculated as described in the legend of Fig. 2B. Data were showed as the averages of n≥3 experiments in which HEK293 cells pretreated with 100nM DAMGO (panel A) and 100nM morphine (panel B) respectively to induce OPRM1 desensitization. * denotes p < 0.05; and ** denotes p< 0.01.

Fig. 6. Effect of βArr2 overexpression on DAMGO- and morphine-induced OPRM1 desensitization in HEK293 cells

The abilities of DAMGO to induce the desensitization of wild type OPRM1 and phosphorylation deficient OPRM1(363/370/375) were examined in HEK293 cells transfected with βArr2-FLAG (○) or with mock transfection (■). Cells were pretreated with 100nM DAMGO or morphine for the indicated time. The agonist-induced desensitization rate was calculated as described in the legend of Fig. 2B. Data were showed as the averages of n≥3 experiments in which wild type OPRM1 was pretreated with 100nM DAMGO (panel A) and 100nM morphine (panel C) or OPRM1(363/370/375) was pretreated 100nM DAMGO (panel B) and 100nM morphine (panel D) respectively. * denotes p < 0.05; and ** denotes p< 0.01.

Fig. 7. Effect of βArr2 depletion on DAMGO- and morphine-induced OPRM1 desensitization in MEF cells

Wild type, βArr2^−/− and βArr1/2^−/− MEF cells were infected with adenovirus containing OPRM1 and OPRM1 expressing level was monitored by receptor binding assay as described in experiment procedures. The abilities of 100nM DAMGO and morphine to induce the desensitization of OPRM1 in wild type MEF cells (■) βArr1/2^−/− MEF cells (○) and βArr2^−/− MEF cells (▲) was examined. Cells were pretreated with 100nM DAMGO or morphine, and agonist-induced desensitization was obtained as described in legend of Figure 2. Data summarize the average of n≥3 experiments in which the HEK293 cells were treated with 100nM DAMGO (panel A) and 100nM morphine (panel B) for various time to induce OPRM1 desensitization. *denotes p < 0.05 and ** denotes p< 0.01.