Ketamine Improves Desensitization of µ-Opioid Receptors Induced by Repeated Treatment with Fentanyl but Not with Morphine

Abstract

The issue of tolerance to continuous or repeated administration of opioids should be addressed. The ability of ketamine to improve opioid tolerance has been reported in clinical studies, and its mechanism of tolerance may involve improved desensitization of μ-opioid receptors (MORs). We measured changes in MOR activity and intracellular signaling induced by repeated fentanyl and morphine administration and investigated the effects of ketamine on these changes with human embryonic kidney 293 cells expressing MOR using the CellKey™, cADDis cyclic adenosine monophosphate, and PathHunter^® β-arrestin recruitment assays. Repeated administration of fentanyl or morphine suppressed the second MOR responses. Administration of ketamine before a second application of opioids within clinical concentrations improved acute desensitization and enhanced β-arrestin recruitment elicited by fentanyl but not by morphine. The effects of ketamine on fentanyl were suppressed by co-treatment with an inhibitor of G-protein-coupled receptor kinase (GRK). Ketamine may potentially reduce fentanyl tolerance but not that of morphine through modulation of GRK-mediated pathways, possibly changing the conformational changes of β-arrestin to MOR.

Keywords: G protein receptor kinase; desensitization; fentanyl; ketamine; morphine; tolerance; µ-opioid receptor; β-arrestin.

Figure 1

Changes in MOR activity with repeated administration of fentanyl or morphine using the CellKey™ assay. The cells expressing MOR were treated with fentanyl or morphine (first administration) for 25 min. After washing and incubation for 30 min, the same dose of each opioid was administered (second administration) and cellular impedance was measured (a). Changes in impedance (ΔZiec) with repeated administration of 1–1000 nM fentanyl (b) and 10–10,000 nM morphine (c) (two-way ANOVA followed by post hoc Tukey’s test). All data are presented as means ± standard error of mean (SEM) (n = 6–12). **** p < 0.0001; ns—not significant; V—vehicle; Fen—fentanyl; Mrp—morphine.

Figure 2

Effects of ketamine on MOR activity induced by single or second administration of fentanyl or morphine in MOR-expressing cells using the CellKey™ assay. Pretreatment with ketamine on single administration of fentanyl or morphine; 100 µM ketamine was pretreated for 30 min before a single administration of fentanyl or morphine (a). Effects of pretreatment with ketamine on changes in impedance (ΔZiec) induced by single administration (first administration) of 1–1000 nM fentanyl (b) or 10–10,000 nM morphine (c) (two-way ANOVA followed by post hoc Tukey’s test). Intermediate treatment with ketamine on repeated administration of fentanyl or morphine; ketamine (1–100 µM) was administered for 30 min before the second administration of fentanyl or morphine (d). Effects of intermediate treatment with ketamine on changes in impedance induced by repeated administration of fentanyl at doses of 10 nM (e), 100 nM (f), 1000 nM (g), and 10,000 nM morphine (h) (one-way ANOVA followed by post hoc Tukey’s test in comparison to the vehicle to fentanyl or vehicle to morphine groups). All data are presented as means ± SEM (n = 6–12). * p < 0.05; ** p < 0.01; **** p < 0.0001; ns—not significant; V—vehicle; Fen—fentanyl; Mrp—morphine; Ket—ketamine.

Figure 3

Effects of MK-801 on the decrease in MOR activity caused by repeated administration of fentanyl and intracellular signal inhibitors on ketamine-induced decrease in MOR activity caused by repeated administration of fentanyl in MOR-expressing cells using the CellKey™ assay. MK-801 (1–100 µM) was administered for 30 min before the second administration of fentanyl (a). Effects of intermediate treatment of 1–100 µM MK-801 on changes in impedance (Δziec) with repeated administration of 100 nM fentanyl (b) (one-way ANOVA followed by post hoc Tukey’s test in comparison to the vehicle to fentanyl group). Each inhibitor was administered concurrently with ketamine (c). Effects of impedance in intermediate treatment of CMPD101 (d), U0126 (e), Ro 31-8220 (f), or JNK inhibitor II (g) at doses of 0.001–10 µM with 100 µM ketamine on impedance induced by repeated administration of 100 nM fentanyl (one-way ANOVA followed by post hoc Tukey’s test in comparison to the ketamine pretreatment before the second administration of fentanyl group). All data are presented as means ± SEM (n = 6–12). * p < 0.05; **** p < 0.0001; ns—not significant; V—vehicle; Fen—100 nM fentanyl; Ket—100 µM ketamine.

Figure 4

Changes in decrease in intracellular cAMP induced by repeated administration of fentanyl or morphine and effects of intermediate treatment of ketamine on the rescue in intracellular cAMP induced by repeated administration of fentanyl or morphine in MOR-expressing cells using cADDis cAMP assay. Changes in intracellular cAMP with repeated administration at the same dose of 1–1000 nM fentanyl (a) and 10–10,000 nM morphine (b) (two-way ANOVA followed by post hoc Tukey’s test). Effects of intermediate treatment with 10–100 µM ketamine on the rescue of intracellular cAMP induced by repeated administration of fentanyl at doses of 10 nM (c), 100 nM (d), and 10,000 nM morphine (e) (one-way ANOVA followed by post hoc Tukey’s test in comparison to the vehicle to fentanyl or vehicle to morphine groups). All data are presented as means ± SEM (n = 6). * p < 0.05; ** p < 0.01; **** p < 0.0001; ns—not significant; V—vehicle; Fen—fentanyl; Mrp—morphine.

Figure 5

Effects of intracellular signal inhibitors on the rescue of intracellular cAMP induced by repeated administration of fentanyl with ketamine in MOR-expressing cells using cADDis cAMP assay. Effects of 0.01–10 µM CMPD101 (a) or U0126 (b) on the rescue of intracellular cAMP induced by repeated administration of 100 nM fentanyl with 100 µM ketamine (one-way ANOVA followed by post hoc Tukey’s test in comparison to the ketamine pretreatment before the second administration of fentanyl group). All data are presented as means ± SEM (n = 6). * p < 0.05; **** p < 0.0001; ns—not significant; V—vehicle; Fen—100 nM fentanyl; Ket—100 µM ketamine.

Figure 6

Effects of intermediate treatment with ketamine on changes in β-arrestin recruitment levels caused by repeated administration of fentanyl or morphine in MOR-expressing cells using the PathHunter^® eXpress β-arrestin assay. Effects of intermediate treatment with 10–100 µM ketamine on changes in β-arrestin recruitment levels caused by repeated administration of fentanyl at the same doses of 10 nM (a), 100 nM (b), and 10,000 nM morphine (c) (one-way ANOVA followed by post hoc Tukey’s test in comparison to the repeated administration of fentanyl or morphine groups). All data are presented as means ± SEM (n = 6). ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns—not significant; V—vehicle; Fen—fentanyl; Mrp—morphine; Ket—ketamine.

Figure 7

Effects of intracellular signal inhibitors on changes in β-arrestin recruitment levels to MOR induced by repeated administration of fentanyl with ketamine in MOR-expressing cells using the PathHunter^® eXpress β-arrestin assay. Effects of 0.01–10 µM of CMPD101 (a) or U0126 (b) on changes in β-arrestin recruitment to MOR induced by repeated administration of 100 nM fentanyl with 100 µM ketamine (one-way ANOVA followed by post hoc Tukey’s test in comparison to the ketamine pretreatment before the second administration of fentanyl group). All data are presented as SEM (n = 6). * p < 0.05; **** p < 0.0001; ns—not significant; V—vehicle; Fen—100 nM fentanyl; Ket—100 µM ketamine.