Endothelin-converting enzyme 2 differentially regulates opioid receptor activity

Abstract

Background and purpose: Opioid receptor function is modulated by post-activation events such as receptor endocytosis, recycling and/or degradation. While it is generally understood that the peptide ligand gets co-endocytosed with the receptor, relatively few studies have investigated the role of the endocytosed peptide and peptide processing enzymes in regulating receptor function. In this study, we focused on endothelin-converting enzyme 2 (ECE2), a member of the neprilysin family of metallopeptidases that exhibits an acidic pH optimum, localizes to an intracellular compartment and selectively processes neuropeptides including opioid peptides in vitro, and examined its role in modulating μ receptor recycling and resensitization.

Experimental approach: The effect of ECE2 inhibition on hydrolysis of the endocytosed peptide was examined using thin-layer chromatography and on μ opioid receptor trafficking using either elisa or microscopy. The effect of ECE2 inhibition on receptor signalling was measured using a cAMP assay and, in vivo, on antinociception induced by intrathecally administered opioids by the tail-flick assay.

Key results: The highly selective ECE2 inhibitor, S136492, significantly impaired μ receptor recycling and signalling by only those ligands that are ECE2 substrates and this was seen both in heterologous cells and in cells endogenously co-expressing μ receptors with ECE2. We also found that ECE2 inhibition attenuated antinociception mediated only by opioid peptides that are ECE2 substrates.

Conclusions and implications: These results suggest that ECE2, by selectively processing endogenous opioid peptides in the endocytic compartment, plays a role in modulating opioid receptor activity.

Linked articles: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

Figure 1

ECE2 expression leads to enhanced recycling of μ opioid receptors. CHO-μ or CHO-μ-ECE2 cells were incubated with 1 μM DAMGO or 100 nM dynorphin B (Dyn B) for 30 min (t = 0); cells were washed and incubated for either 120 min (A) or the indicated time periods (B) without the agonist. Cell surface receptors were quantified by elisa as described in Methods. Levels of cell surface receptors before agonist treatment were taken as 100% for each individual experiment. % recycled receptors were calculated by subtracting surface receptors at t = 0 (30 min internalization) from each recycling time point. The data represent mean ± SEM from four to five independent experiments carried out in quadruplicate. ^***P < 0.001, Student's t-test.

Figure 2

Selectivity of the inhibitor S136492 for ECE2. (A, B) Purified ECE2 (A) or ECE1 (B) was assayed for 10 min in the absence or presence of S136492 (20 μM) at pH 5.5 or pH 7.4 using the synthetic quenched fluorescent substrate, McaBk2, as described previously (Gagnidze et al., 2008). Relative fluorescence units before the addition of substrate (i.e. t = 0) were taken as 100%. (C) The IC₅₀ values were derived by carrying out enzymatic assays in the absence or presence of SM136492 (0–50 μM) at pH 5.5 or pH 7.4 as described previously (Gagnidze et al., 2008). Data represent mean ± SEM of three independent experiments in triplicate. (D, E) Solubilized midbrain preparations from wild-type (D) and ECE2 knockout mice (ECE2 k/o) (E) were assayed for their enzymatic activity for 10 min using the synthetic quenched fluorescent substrate, McaBk2, in the absence or presence of S136492 (20 μM) at pH 5.5 or pH 7.4 as described previously (Gagnidze et al., 2008). Relative fluorescence units before the addition of substrate (i.e. t = 0) were taken as 100%. Data represent mean ± SEM from three animals per group in triplicate.

Figure 3

ECE2 activity is required for modulating the recycling but not the internalization of μ opioid receptors. (A) Receptors in CHO-μ-ECE2 cells were pre-labelled with anti-FLAG antibody (1 μg) for 60 min at 4°C (t = 0) as described in Methods. Pre-labelled cells were incubated with DAMGO (1 μM) or dynorphin B (Dyn B, 100 nM) for 30 min to allow for receptor internalization without (Int) or with 20 μM S136492 (Int + ECE2 inh). The level of surface receptors determined by elisa at t = 0 for each individual experiment was taken as 100 % surface receptors. % internalized receptors were calculated as 100 – % surface receptors after agonist treatment. (B) CHO-μ-ECE2 cells were incubated with DAMGO (1 μM) or dynorphin B (100 nM) for 30 min (t = 0); cells were washed and incubated for 60 min in medium without (Rec) or with 20 μM S136492 (Rec + ECE2 inh). (C) CHO-μ-ECE2 cells were incubated with DAMGO (1 μM) or dynorphin B (100 nM) for 30 min (t = 0); cells were washed and incubated for 60 min in medium containing 0–50 μM S136492 (ECE2 inh). Cell surface receptors were quantified by elisa as described in Methods. The level of surface receptors before agonist treatment for each individual experiment was taken as 100%. % recycled receptors (B, C) were calculated by subtracting % of surface receptors at t = 0 (30 min internalization) from each recycling time point. Data represent mean ± SEM of five independent experiments in triplicate. ^***P < 0.001; Student's t-test.

Figure 4

ECE2 and an acidic pH are required for modulation of μ opioid receptor recycling following endocytosis by agonists that are ECE2 substrates. CHO-μ-ECE2 cells were incubated with DAMGO (1 μM) (A, B) or dynorphin B (Dyn B, 100 nM) (C, D) for 30 min (t = 0); cells were washed and incubated for 60 min with medium without (control) or with 20 μM ECE2 inhibitor (+S136492), 10 μM phosphoramidon (+Phosphor), 100 μM chloroquine, 100 nM bafilomycin, 10 μM captopril, 10 μM thiorphan or 100 nM cycloheximide (+Cyclohex). Cell surface receptors were quantified by elisa as described in Methods. % recycled receptors were calculated as described in legend to Figure 1 and % recycled receptors obtained with control were expressed as 100%. Data represent mean ± SEM of five to eight independent experiments in triplicate. ^***P < 0.001; one-way anova.

Figure 5

ECE2 co-localizes with μ opioid receptors in intracellular compartments and cleaves [³H]-DAMGO at acidic pH. (A) Co-localization of ECE2 with EEA1, a marker for early endosomes. CHO-μ-ECE2 cells were stained with antibodies to the HA tag on ECE2 or to EEA1 and staining visualized by microscopy as described in Methods. (B) Co-localization of ECE2 with μ opioid receptors in intracellular compartments. CHO-μ-ECE2 cells were stained with antibodies to FLAG or HA tags and staining visualized by microscopy as described in Methods. (C) [³H]-DAMGO (10 nM) was incubated without (C) or with purified ECE2 (D, E) at pH 5.5 for 30 min in the absence (D) or presence of 20 μM S136492 (E) followed by thin-layer chromatographic analysis as described in Methods. (F) CHO-μ-ECE2 cells were incubated with 10 nM [³H]-DAMGO for 30 min at 37°C without or with either 20 μM S136492, 10 μM captopril or 100 μM chloroquine. The cells were subjected to acid wash to remove surface bound radiolabel, lysed, and subjected to thin-layer chromatographic analysis as described in Methods. The arrows indicate the position of full-length [³H]-DAMGO. Data in (C–F) represent mean ± SEM of four to six independent experiments.

Figure 6

Recycling of receptors endocytosed by a peptidic agonist (and not by a non-peptidic agonist) is blocked by ECE2 inhibition. CHO-μ-ECE2 cells were incubated with DAMGO (1 μM) (A), dynorphin B (Dyn B, 100 nM) (B) or fentanyl (1 μM) (C) for 30 min (t = 0); cells were washed and incubated for various time periods in medium without (control) or with 20 μM S136492 (+ECE2 inh). Cell surface receptors were quantified by elisa as described in Methods. Levels of cell surface receptors before agonist treatment for each individual experiment were taken as 100%. % recycled receptors were calculated by subtracting surface receptors at t = 0 (30 min internalization) from each recycling time point. Data represent mean ± SEM of five to eight independent experiments in triplicate.

Figure 7

ECE2 inhibition prevents recycling of μ opioid receptors endocytosed following exposure to endogenous peptides that are the substrates of ECE2. CHO-μ-ECE2 cells were incubated with either DAMGO (1 μM), dynorphin B (Dyn B, 100 nM), BAM22 (100 nM), [Leu]enkephalin (100 nM) or fentanyl (1 μM) for 30 min (t = 0); cells were washed and incubated for various time periods in medium without (control) or with 20 μM S136492 (+ECE2 inh). Cell surface receptors were quantified by elisa as described in Methods. Levels of cell surface receptors before agonist treatment for each individual experiment were taken as 100%. % recycled receptors were calculated by subtracting surface receptors at t = 0 (30 min internalization) from each recycling time point and expressing % recycled receptors obtained with control as 100. Data represent mean ± SEM of five independent experiments in triplicate. ^***P < 0.001; ns, not significant; Student's t-test.

Figure 8

Inhibition of ECE2 activity affects μ opioid receptor resensitization. CHO-μ-ECE2 cells were incubated with DAMGO (1 μM) (A), dynorphin B (Dyn B, 100 nM) (B) or fentanyl (1 μM) (C) along with 10 μM forskolin for 20 min; cells were then washed and incubated for 60 min (recycling phase) in medium without or with 20 μM S136492 (ECE2 inh). Cells were washed and cAMP levels in response to a second 5 min treatment with DAMGO, dynorphin B or fentanyl were determined using the DiscoveRx kit as described in Methods. Data represent mean ± SEM of five independent experiments in sixtuplicate. RLU, relative luminescence units; ^***P < 0.001; ns, not significant; Student's t-test.

Figure 9

Co-localization of ECE2 and μ opioid receptors in F11 cells and DRGs. (A) qRT-PCR analysis shows that F11 cells, small and large DRGs, express ECE2 mRNA. Data represent mean ± SEM (n = 3–6). ***P < 0.001. (B) Co-localization of ECE2 with EEA1, a marker for early endosomes. F11 cells were stained with antibodies to ECE2 or EEA1 and staining was visualized by microscopy as described in Methods. There is substantial co-localization of ECE2 (red) with EEA1 (green). (C) Co-localization of ECE2 with μ opioid receptors in intracellular compartments. F11 cells were stained with antibodies to μ receptors and to ECE2, and staining was visualized by microscopy as described Methods. Pearson's coefficient shows that during receptor internalization (Int; agonist treatment for 30 min), there is an increase in co-localization of μ opioid receptors (green) with ECE2 (red), which is reduced during recycling with media (Rec) but not following inhibition of ECE2 activity during recycling (Rec + ECE2 inh). n = 10 fields (10–15 cells/field). *P < 0.05; ^**P < 0.01; ^***P < 0.001; Student's t-test.

Figure 10

Inhibition of endogenous ECE2 activity in F11 DRG-derived cell line impairs recycling of native μ opioid receptors. F11 cells were incubated with 1 μM DAMGO (A) or 100 nM dynorphin B (B) for 30 min (t = 0); cells were washed and incubated (0–120 min) in medium without (control) or with 20 μM S136492 (+ECE2 inh). (C) F11 cells were incubated with 1 μM DAMGO, 100 nM of either dynorphin B, or [Leu]enkephalin, for 30 min (t = 0); cells were washed and incubated for 60 min in medium without (control) or with 20 μM S136492 (+ECE2 inh). Cell surface receptors were quantified by elisa as described in Methods. Levels of cell surface receptors before agonist treatment for each individual experiment were taken as 100%. % recycled receptors were calculated by subtracting surface receptors at t = 0 (30 min internalization) from each recycling time point. For (C), % recycled receptors obtained with control were taken as 100. The data represent mean ± SEM of five to eight independent experiments in triplicate. ^***P < 0.001; ns, not significant; Student's t-test.

Figure 11

Inhibition of endogenous ECE2 activity affects native μ opioid receptor resensitization in F11 cells. F11 cells were incubated with DAMGO (1 μM) (A), dynorphin B (Dyn B, 100 nM) (B) or fentanyl (1 μM) (C), along with 10 μM forskolin for 20 min; cells were then washed and incubated for 60 min in medium without or with 20 μM S136492 (ECE2 inh). Cells were washed and cAMP levels in response to a second 5 min treatment with DAMGO, dynorphin B or fentanyl were determined using the DiscoveRx kit as described in Methods. Data represent mean ± SEM of four independent experiments in sixtuplicate. RLU, relative luminescence units; ^***P < 0.001; ns, non-significant; Student's t-test.

Figure 12

ECE2 plays a role in modulating opioid-mediated analgesia. C57BL/6 mice (5–8 per group) were injected (i.t.) with either vehicle (6% DMSO, 5% Tween 80 and protease inhibitor cocktail) or vehicle containing fentanyl (0.3 nmol) (A), [Leu]enkephalin (10 nmol) (B) or dynorphin B (10 nmol) (C) in the absence or presence of S136492 (ECE2 inh, 3 nmol), and analgesia was measured using the tail-flick assay at 5,10, 15, 30, 60 and 90 min. *P < 0.05 as compared with the absence of the ECE2 inhibitor; Student's t-test.