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Perioperative Medicine  |   May 2017
Hypoxia Induces Internalization of κ-Opioid Receptor
Author Notes
  • From the Department of Anesthesia, Beijing Tongren Hospital, Capital Medical University, Beijing, China (C.X., X.L.); Department of Anesthesiology and Critical Care, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (C.X., X.L., C.C., H.B., R.L.); and Department of Anesthesia, Beijing Shijitan Hospital, Capital Medical University, Beijing, China (T.L.).
  • C.X. and X.L. contributed equally to this article.
    C.X. and X.L. contributed equally to this article.×
  • Submitted for publication May 23, 2016. Accepted for publication January 17, 2017.
    Submitted for publication May 23, 2016. Accepted for publication January 17, 2017.×
  • Address correspondence to Dr. Liu: Department of Anesthesiology and Critical Care, Perelman School of Medicine, University of Pennsylvania, 336 John Morgan Building, 3620 Hamilton Walk, Philadelphia, Pennsylvania 19104. liur@uphs.upenn.edu. Information on purchasing reprints may be found at www.anesthesiology.org or on the masthead page at the beginning of this issue. Anesthesiology’s articles are made freely accessible to all readers, for personal use only, 6 months from the cover date of the issue.
Article Information
Perioperative Medicine / Basic Science / Pain Medicine / Pharmacology
Perioperative Medicine   |   May 2017
Hypoxia Induces Internalization of κ-Opioid Receptor
Anesthesiology 5 2017, Vol.126, 842-854. doi:10.1097/ALN.0000000000001571
Anesthesiology 5 2017, Vol.126, 842-854. doi:10.1097/ALN.0000000000001571
Abstract

Background: It has been demonstrated that κ-opioid receptor agonists can reduce hypoxia–ischemia brain injury in animal models. However, it is unclear how the κ-opioid receptor responds to hypoxia–ischemia. In the current study, the authors used an in vitro model of oxygen–glucose deprivation and reoxygenation to explore how κ-opioid receptors respond to hypoxia and reoxygenation.

Methods: Mouse neuroblastoma Neuro2A cells were stably transfected with mouse κ-opioid receptor–tdTomato fusion protein or Flag-tagged mouse κ-opioid receptor, divided into several groups (n = 6 to 12), and used to investigate the κ-opioid receptor movement. Observations were performed under normal oxygen, at 30 min to 1 h after oxygen–glucose deprivation and at 1 h after reoxygenation using high-resolution imaging techniques including immunoelectronmicroscopy in the presence and absence of κ-opioid receptor antagonist, dynamin inhibitors, potassium channel blockers, and dopamine receptor inhibitor.

Results: Hypoxic conditions caused the κ-opioid receptor to be internalized into the cells. Inhibition of dynamin by Dyngo-4a prevented the receptor internalization. Interestingly, a specific κ-opioid receptor antagonist norbinaltorphimine blocked internalization, suggesting the involvement of activation of a specific κ-opioid receptor. κ-Opioid receptor internalization appears to be reversed by reoxygenation. Quantities of intracellular κ-opioid receptor-associated gold particles as demonstrated by immunoelectron microscopy were increased from 37 to 85% (P < 0.01) after oxygen–glucose deprivation. Potassium channel blockers and dopamine receptor inhibitor failed to block hypoxia-induced κ-opioid receptor internalization.

Conclusions: Hypoxia induces reversible κ-opioid receptor internalization, which was inhibited by selective κ-opioid receptor antagonists or dynamin inhibitor, and can be reversed by reoxygenation in neuroblastoma cells, indicating the modulating effects between κ-opioid receptor and hypoxia via κ-opioid receptor activation and the dynamin-dependent mechanism.

What We Already Know about This Topic
  • Opioid receptors belong to the G-protein–coupled receptor family, internalization of which occurs in response to agonist exposure

  • Agonist-induced internalization of the κ-opioid receptor is mediated through G-protein–coupled receptor kinase 2-, dynamin-, and β-arrestin–dependent mechanisms

  • κ-Opioid receptor agonists have neuroprotective effects in animal hypoxic–ischemic injury models

What This Article Tells Us That Is New
  • While most κ-opioid receptors are located on the cell membrane of mouse Neuro2A neuroblastoma cells, oxygen–glucose deprivation induced significant internalization of them

  • Oxygen–glucose deprivation-induced internalization of κ-opioid receptors can be blocked by specific κ-opioid receptor antagonists or a dynamin inhibitor and can be reversed by reoxygenation

THE opioid receptors belong to the G-protein–coupled receptor (GPCR) family with seven transmembrane domains.1  The opioid system is composed of three major receptor subtypes, μ-opioid receptor (MOR), κ-opioid receptor (KOR), and δ-opioid receptor (DOR), and is essentially an inhibitory neurotransmitter system. The receptors are expressed not only within the central nervous system2–5  but also on peripheral sensory nerve terminals.6,7  The opioid system is involved in the modulation of various physiologic and pathologic activities, including nociceptive–analgesic signal transduction, neurotransmitter secretion, respiration, immune responses, as well as ion channel functions.
GPCR internalization is one of the important processes in response to agonist exposure, which occurs over a timescale ranging from minutes to hours.8–10  Agonist-induced KOR internalization has been extensively studied.11  It was demonstrated that agonist-induced internalization of the human KOR is required for down-regulation.12  Previous in vitro studies have shown that internalization of KOR is agonist specific and occurs in a dose- and time-dependent manner.13–18  For example, KOR is internalized by the agonists U50,488 and dynorphin 1–17 but not other agonists such as etorphine or levorphanol.17  KOR antagonist norbinaltorphimine, GPCR phosphorylation inhibitor phosducin, as well as endocytosis-blocking agents strongly inhibited selective KOR agonist U50,488H-induced internalization of KOR.14,18,19  Cells have been shown to mediate agonist-induced internalization of KOR through GPCR kinase 2-, dynamin-, and β-arrestin–dependent mechanisms.14,20,21  Although internalization of GPCRs is generally described as a rapid agonist-induced motion of the receptor into the cytoplasm from the cell membrane,14  long-term agonist exposure leads to the down-regulation of the receptors trafficked to the lysosomes and proteasomes for degradation.12 
Recently, it has been shown that KOR agonists exhibit neuroprotective effects in animal models of hypoxic– ischemic injury.22–24  Considering the involvement of KOR in hypoxic–ischemic, it is of great importance to investigate the effect of hypoxic conditions on the cellular translocation of KOR. In this study, we explored whether hypoxic conditions induce KOR internalization in agonistic fashion. We used mouse Neuro2A cells stably expressing mouse KOR (mKOR)–tdTomato fusion protein or Flag-tagged mKOR (mKOR-FLAG) to simulate hypoxic–ischemic injury in vitro and track the KOR under oxygen–glucose deprivation (OGD) conditions.
Materials and Methods
M1 mouse anti-FLAG monoclonal antibody, normal goat serum, poly-d-lysine, Triton X-100, norbinaltorphimine, glibenclamide, iberiotoxin, and sulpiride were obtained from Sigma-Aldrich (USA). Salvinorin A (purity greater than 98%) was obtained from Apple Pharms (USA). Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin G (IgG), Alexa Fluor 488-conjugated goat anti-rabbit IgG with 10-nm colloidal gold conjugate, antifade mounting medium with 4',6-diamidino-2-phenylindole (DAPI), minimum essential medium (MEM) powder, glucose-free Dulbecco modified Eagle medium (DMEM), and fetal bovine serum were obtained from Invitrogen (USA). Anti-red fluorescent protein antibody and Dyngo-4a were obtained from Abcam (USA). Geneticin was obtained from Cellgro Mediatech (USA). Lab-TekΠSlide Chamber was obtained from Thermo Scientific (USA). Hypoxia system with gas mixture (95% N2, 5% CO2) was obtained from Airgas USA, LLC (USA). All other chemicals used were of reagent grade and obtained from Sigma-Aldrich.
Cell Lines
Mouse neuroblastoma Neuro2A cells stably transfected with mKOR–tdTomato fusion protein (N2A-mKOR-tdT) and Flag-tagged mKOR (N2A-mKOR-FLAG) cells were prepared as described previously.25–27  Briefly, the red fluorescent protein tdTomato was fused to the C-terminus and the FLAG epitope was tagged to the N-terminus of the mKOR to make mKOR-tdT and mKOR-FLAG fusion proteins. Next, mKOR-tdT and mKOR-FLAG were separately transfected and expressed in Neuro2A cells. Cells were cultured in MEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. Geneticin (0.2%) and Blasticidin (0.005%; Sigma-Aldrich, USA) were used for the selection of N2A-mKOR-tdT and N2A-mKOR-FLAG, respectively. Cells were kept in a humidified atmosphere consisting of 95% air and 5% CO2 at 37°.
OGD
To simulate OGD, cells were washed twice with the glucose-free DMEM, incubated in a plexiglass chamber (Billups-Rothenberg, Inc., USA), and flushed with hypoxic gas mixture for 5 min at the flow rate of 20 l/min. The glass chamber was then sealed and disconnected from the gas source.28,29  Partial pressures of oxygen and carbon dioxide of OGD medium were measured by a blood gas analyzer (RapidLab@248; Siemens Diagnostics, USA).
Cell Viability Assay
3-(4,5-Dimethylthia-2ol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) colorimetric assay was used to assess cell viability (CV). N2A-mKOR-tdT and N2A-mKOR-FLAG cells were seeded (4 × 104 cells/well) in a 96-well plate and cultured overnight. Cells were then incubated in normal growth conditions (control group) or OGD conditions (OGD group) for 0.5, 1, 2, 4, and 8 h. After each treatment, cells were returned to normal growth conditions and incubated with MTT in a final concentration of 0.5 mg/ml for 4 h at 37°. Then, the medium was removed, and the cells were incubated with 10% sodium dodecyl sulfate in 1 mM HCl for 4 h at 37°. Optical density (OD) of each well was measured at 570 nm using a microplate reader (Multiscan MK3; Thermo Labsystems, USA). CV (%) was calculated as the ratio of the OD of individual treatment to the OD of the control group.
OGD-induced KOR Trafficking by Fluorescence and Confocal Microscopy
N2A-mKOR-tdT or N2A-mKOR-FLAG cells were seeded to poly-d-lysine–coated eight-well–chambered cover glasses (Thermo Scientific, USA) and cultured to 80 to 90% confluence. Both cells were divided into three groups: control group, OGD group, and reoxygenation group. For N2A-mKOR-tdT cells in the control group, cells were washed twice with serum-free MEM and incubated in normal growth medium containing glucose in humidified atmosphere at 37°, 95% air, and 5% CO2 for 1 h. Concurrently, in the OGD group, cells were washed twice with the glucose-free DMEM and then incubated under OGD conditions for 0.5 or 1 h. Cells in the reoxygenation group were preincubated under OGD conditions for 1 h before transferring to the normal growth medium containing glucose and then incubated in humidified atmosphere at 37°, 95% air, and 5% CO2 for 1 h for reoxygenation. After each procedure, cells were washed three times with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 20 min at room temperature. Fixed cells were then washed with PBS and mounted using antifade mounting medium with DAPI. Images of mKOR-tdT were acquired using an inverted fluorescence microscope with a 40× objective (Olympus IX70; Olympus, USA) through a Texas Red channel (excitation, 560 nm; emission, 620 nm). Nuclear images were captured through a DAPI channel (excitation, 400 nm; emission, 460 nm).
N2A-mKOR-FLAG cells were used as well-established reference for the detection of KOR trafficking and were prepared for immunofluorescence staining as described previously.17,26  Briefly, M1 mouse anti-FLAG monoclonal antibody was diluted in the MEM (4 μg/ml) containing 0.05% Triton X-100, 4% normal goat serum, and 1 mM CaCl2 marked as feeding medium. N2A-mKOR-FLAG cells were washed twice with the fetal bovine serum-free MEM and then incubated in the feeding medium at 37° for 1 h. Subsequently, N2A-mKOR-FLAG cells were cultured in normal growth medium, OGD conditions for 0.5 to 1 h, or OGD conditions for 1 h followed by 1 h reoxygenation in normal growth medium. Then, N2A-mKOR-FLAG cells were washed with ice-cold Hanks Balanced Salt Solution, fixed with 4% paraformaldehyde, and washed for 5 min with Tris-HCl buffer solution at room temperature. Next, cells were incubated with 2 µg/ml Alexa Fluor 488–conjugated goat anti-mouse IgG (H + L) in 0.05% Triton X-100, 4% normal goat serum, or PBS for 30 min. After washing three times with PBS, cells were mounted, and images of mKOR-FLAG were acquired through fluorescein isothiobyanate channel with excitation at 490 nm and emission at 520 nm.
N2A-mKOR-tdT cells pretreated with norbinaltorphimine (a selective KOR antagonist), Dyngo-4a, glibenclamide (an adenosine triphosphate-sensitive K+ channel inhibitor), iberiotoxin (a selective high-conductance calcium-activated K+ channel inhibitor), or sulpiride (a dopamine D2 receptor antagonist) for 0.5 h were used to investigate whether they could block the OGD-related KOR trafficking.
To discriminate whether the redistributed KOR aggregated in the cell-surface layer or moved into the cytoplasm, N2A-mKOR-tdT and N2A-mKOR-FLAG cells were fixed or stained with immunofluorescence as described above and observed by a confocal microscopy using a 63× oil immersion objective (Leica TCS SP6; Germany). RsRed channel or fluorescein isothiobyanate channel was selected for the observation of N2A-mKOR-tdT or N2A-mKOR-FLAG cells, respectively. A z-stack was performed with a z-step size of 10 μm. For each experiment, cells were observed under the same conditions at least three times in each group.
Agonist-induced KOR Trafficking by Fluorescence Microscopy
To visualize the agonist-induced KOR trafficking, cells treated with 10 μM salvinorin A for 30 min were used to demonstrate the agonist-induced KOR internalization. Cells pretreated with norbinaltorphimine and Dyngo-4a were used to demonstrate the inhibition of the agonist-induced KOR internalization. Fluorescence microscopy was performed as described in the “OGD-induced KOR Trafficking by Fluorescence and Confocal Microscopy” section. For each experiment, cells were observed under the same conditions at least three times in each group.
OGD-induced KOR Trafficking by Single-cell Imaging Flow Cytometry
N2A-mKOR-tdT cells were prepared as a single-cell suspension at 107 cells/ml concentration. After 1-h treatment with or without OGD, circularity feature of mKOR-tdT was captured by ImageStreamX imaging flow cytometer (Amnis, USA) and analyzed by the IDEAS software (EMD Millipore, USA). The circularity feature, which is given by the circularity index, measures the degree of the mKOR-tdT’s deviation from a circle. Its measurement is based on the average distance of the object boundary from its center divided by the variation of this distance. Thus, the closer the object to a circle, the higher the feature value. The circularity and contrast were used to determine the trafficking of mKOR-tdT.
OGD-induced KOR Trafficking Located by Immunoelectron Microscopy
For electron microscopy, N2A-mKOR-tdT cells were cultured in T25 flasks and divided into the control group without OGD and the OGD group receiving the 1-h OGD treatment as described in the “OGD-induced KOR Trafficking by Fluorescence and Confocal Microscopy” section. After 20-min fixation with 4% paraformaldehyde or NaAc (pH = 7.3) at room temperature, the cells were gently scraped, centrifuged with the speed of 1,200 revolutions/min for 5 min, and subjected to −80° by a high-pressure freezing machine with a working pressure of 2,300 bar (HPM 010; Boeckeler Instruments, USA). Cells were then embedded in hydrophilic resin medium HM20 and polymerized with ultraviolet light. Ultrathin sections (80 nm) were cut from the resin-embedded blocks at room temperature by a diamond knife and collected onto 300-mesh nickel grids. Grids were rehydrated with PBS for 10 min; blocked with 1% BSA, 2% normal goat serum, or PBS for 30 min; and incubated with the anti-red fluorescent protein primary antibody (1:500) at room temperature. Sections incubated in 1% BSA or PBS were used as a negative control. The sections were then washed with PBS, incubated with 1% BSA or PBS for 20 min, and immunostained with Alexa Fluor 488-conjugated goat anti-rabbit IgG with 10-nm colloidal gold conjugate for 40 min in a humidified container. Grids were washed with PBS four times, 5 min each, in porcelin plate wells and fixed with 2% glutaraldehyde or PBS. Next, grids were washed with aqua dest three times, 5 min each, and briefly stained with uranyl acetate and lead citrate. Finally, grids were dried, randomly assigned, and examined in a double-blind fashion using a Jeol-JEM100CX electron microscope (JEOL Ltd., Japan) at 60 kV. KOR internalization in various groups studied was quantified by calculating the ratio of gold particles in cytoplasm to the total gold particles for each untrasection with a remarkable nucleus.
Statistical Analysis
All data are shown as mean ± SD unless otherwise indicated. Sample sizes were estimated based on our previous laboratory experience. Statistical significance among the groups was determined by Student’s two-tailed t test or one-way ANOVA followed by the Tukey test. P < 0.05 was considered as statistically significant. Cell cultures were randomly assigned to the experimental groups using random numbers and processed identically throughout the whole experiments. Statistical analyses were performed with Graph-Pad Prism (version 6.01; GraphPad Software, Inc., USA). Images were analyzed using ImageJ (1.48v; National Institutes of Health, USA). All valid data were included in the analysis, without excluding outliers unless otherwise stated.
Results
OGD and CV
The partial pressure of oxygen in the OGD group of N2A-mKOR-tdT cells was significantly lower than that in the control group. There was no statistically significant difference among the OGD groups (fig. 1A). No significant difference was observed in partial pressure of carbon dioxide among groups of N2A-mKOR-tdT cells (fig. 1B). There were no significant CV changes within 2 h of OGD applied to N2A-mKOR-tdT (fig. 1C) or N2A-mKOR-FLAG (fig. 1D), indicating that OGD within 2 h was suitable for the investigation of hypoxia-induced KOR trafficking. Significant cell death was observed only after 4 h of OGD (P < 0.001; fig. 1, C and D).
Fig. 1.
Oxygen–glucose deprivation (OGD) and cell viability. (A) All OGD groups of Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT) cells maintained with 95% N2 and 5% CO2 from 0.5 to 12 h had significantly decreased partial pressure of oxygen (mmHg) in culture medium compared with that in the control group without OGD (n = 6 per group). (B) There was no significant difference among partial pressure of carbon dioxide (mmHg) in culture medium with OGD of N2A-mKOR-tdT cells from 0.5 to 12 h (n = 6 per group). OGD-induced time-dependent decrease of cell viability in both (C) N2A-mKOR-tdT and (D) Neuro2A cells stably transfected with Flag-tagged mouse κ-opioid receptor (N2A-mKOR-FLAG) cells after 4 h of treatment (n = 12 per group). Data are shown as mean ± SD. *P < 0.001.
Oxygen–glucose deprivation (OGD) and cell viability. (A) All OGD groups of Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT) cells maintained with 95% N2 and 5% CO2 from 0.5 to 12 h had significantly decreased partial pressure of oxygen (mmHg) in culture medium compared with that in the control group without OGD (n = 6 per group). (B) There was no significant difference among partial pressure of carbon dioxide (mmHg) in culture medium with OGD of N2A-mKOR-tdT cells from 0.5 to 12 h (n = 6 per group). OGD-induced time-dependent decrease of cell viability in both (C) N2A-mKOR-tdT and (D) Neuro2A cells stably transfected with Flag-tagged mouse κ-opioid receptor (N2A-mKOR-FLAG) cells after 4 h of treatment (n = 12 per group). Data are shown as mean ± SD. *P < 0.001.
Fig. 1.
Oxygen–glucose deprivation (OGD) and cell viability. (A) All OGD groups of Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT) cells maintained with 95% N2 and 5% CO2 from 0.5 to 12 h had significantly decreased partial pressure of oxygen (mmHg) in culture medium compared with that in the control group without OGD (n = 6 per group). (B) There was no significant difference among partial pressure of carbon dioxide (mmHg) in culture medium with OGD of N2A-mKOR-tdT cells from 0.5 to 12 h (n = 6 per group). OGD-induced time-dependent decrease of cell viability in both (C) N2A-mKOR-tdT and (D) Neuro2A cells stably transfected with Flag-tagged mouse κ-opioid receptor (N2A-mKOR-FLAG) cells after 4 h of treatment (n = 12 per group). Data are shown as mean ± SD. *P < 0.001.
×
OGD-induced KOR Internalization
Under normoxia conditions, most KORs were located on the cell surface. However, after 30 min of OGD treatment, KOR trafficking was observed in both N2A-mKOR-tdT and N2A-mKOR-FLAG cells. Detected fluorescence signals indicated the KORs moved into the cytoplasm. This phenomenon became prominent with the prolonged hypoxia (fig. 2A–C). After 1 h of reoxygenation, most of the KORs recycled back to the cell surface in both cell lines (fig. 2D). To reconcile internalization of KORs with changes in the receptor trafficking after the OGD treatment, confocal microscopy was performed. A z-stack of an optical section, 10 µm in thickness, was captured, and similar movement of KORs was observed (fig. 3).
Fig. 2.
Oxygen–glucose deprivation (OGD)-induced κ-opioid receptor (KOR) trafficking of Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT) and Flag-tagged mouse κ-opioid receptor (N2A-mKOR-FLAG) cells with a similar pattern. N2A-mKOR-tdT cells expressing mKOR–tdTomato fusion protein (left, red) were either untreated (A) or treated with OGD for 0.5 h (B) or 1 h (C) before the fixation. N2A-mKOR-FLAG cells labeled with anti-FLAG monoclonal antibody were fixed followed by immunostaining with anti-FLAG Alexa Fluor 488-conjugated antibody (right, green). Both mKOR-tdT and mKOR-FLAG were distributed on the cell surface of the untreated group. However, both mKOR-tdT and mKOR-FLAG were internalized as punctuated under OGD. Moreover, OGD-induced change in KOR trafficking could be reversed by reoxygenation (D). The figures represent one of the three independent experiments with similar results. Examples of mKOR-tdT and mKOR-FLAG are indicated by the yellow arrows. Scale bar: AD, 20 μm.
Oxygen–glucose deprivation (OGD)-induced κ-opioid receptor (KOR) trafficking of Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT) and Flag-tagged mouse κ-opioid receptor (N2A-mKOR-FLAG) cells with a similar pattern. N2A-mKOR-tdT cells expressing mKOR–tdTomato fusion protein (left, red) were either untreated (A) or treated with OGD for 0.5 h (B) or 1 h (C) before the fixation. N2A-mKOR-FLAG cells labeled with anti-FLAG monoclonal antibody were fixed followed by immunostaining with anti-FLAG Alexa Fluor 488-conjugated antibody (right, green). Both mKOR-tdT and mKOR-FLAG were distributed on the cell surface of the untreated group. However, both mKOR-tdT and mKOR-FLAG were internalized as punctuated under OGD. Moreover, OGD-induced change in KOR trafficking could be reversed by reoxygenation (D). The figures represent one of the three independent experiments with similar results. Examples of mKOR-tdT and mKOR-FLAG are indicated by the yellow arrows. Scale bar: A–D, 20 μm.
Fig. 2.
Oxygen–glucose deprivation (OGD)-induced κ-opioid receptor (KOR) trafficking of Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT) and Flag-tagged mouse κ-opioid receptor (N2A-mKOR-FLAG) cells with a similar pattern. N2A-mKOR-tdT cells expressing mKOR–tdTomato fusion protein (left, red) were either untreated (A) or treated with OGD for 0.5 h (B) or 1 h (C) before the fixation. N2A-mKOR-FLAG cells labeled with anti-FLAG monoclonal antibody were fixed followed by immunostaining with anti-FLAG Alexa Fluor 488-conjugated antibody (right, green). Both mKOR-tdT and mKOR-FLAG were distributed on the cell surface of the untreated group. However, both mKOR-tdT and mKOR-FLAG were internalized as punctuated under OGD. Moreover, OGD-induced change in KOR trafficking could be reversed by reoxygenation (D). The figures represent one of the three independent experiments with similar results. Examples of mKOR-tdT and mKOR-FLAG are indicated by the yellow arrows. Scale bar: AD, 20 μm.
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Fig. 3.
Oxygen–glucose deprivation (OGD)-induced κ-opioid receptor trafficking was further confirmed by confocal microscopy. Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT; left, red) and Flag-tagged mouse κ-opioid receptor (N2A-mKOR-FLAG) cells (right, green) were either untreated (A) or treated with OGD for 1 h (B) after the fixation and immunostaining as described in figure 2. A single 10-µm layer out of the z-stack is shown. Examples of mKOR-tdT and mKOR-FLAG are indicated by the yellow arrows. Scale bar: A, B, 20 μm.
Oxygen–glucose deprivation (OGD)-induced κ-opioid receptor trafficking was further confirmed by confocal microscopy. Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT; left, red) and Flag-tagged mouse κ-opioid receptor (N2A-mKOR-FLAG) cells (right, green) were either untreated (A) or treated with OGD for 1 h (B) after the fixation and immunostaining as described in figure 2. A single 10-µm layer out of the z-stack is shown. Examples of mKOR-tdT and mKOR-FLAG are indicated by the yellow arrows. Scale bar: A, B, 20 μm.
Fig. 3.
Oxygen–glucose deprivation (OGD)-induced κ-opioid receptor trafficking was further confirmed by confocal microscopy. Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT; left, red) and Flag-tagged mouse κ-opioid receptor (N2A-mKOR-FLAG) cells (right, green) were either untreated (A) or treated with OGD for 1 h (B) after the fixation and immunostaining as described in figure 2. A single 10-µm layer out of the z-stack is shown. Examples of mKOR-tdT and mKOR-FLAG are indicated by the yellow arrows. Scale bar: A, B, 20 μm.
×
Salvinorin A, a selective KOR agonist, induced KOR internalization, which was similar to that of OGD-induced KOR trafficking (fig. 4). Importantly, both Dyngo-4a and norbinaltorphimine inhibited OGD-induced KORs movement as well as that induced by Salvinorin A (fig. 5A), indicating that KOR movement under OGD conditions was an internalization event. Pretreatment with both Dyngo-4a and norbinaltorphimine displayed no toxicity on the N2A-mKOR-tdT cells (fig. 5B).
Fig. 4.
Oxygen–glucose deprivation (OGD)-induced κ-opioid receptor (KOR) trafficking shared similar pattern of KOR agonist-induced KOR internalization. Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT) cells were untreated in control (A), incubated with 1 μM salvinorin A (SA), a selective KOR agonist, for 1 h (B), or treated with OGD for 1 h (C). Cells were fixed, mounted with 4',6-diamidino-2-phenylindole (DAPI), and observed by an inverted fluorescence microscope. SA induced KOR internalization, with a decrease of mKOR-tdT in the cell surface and an increase of crescent-shaped mKOR-tdT in the cytoplasm, which was similar to OGD-induced mKOR-tdT trafficking on the left. Merged images on the right show that both the internalized KORs by SA and OGD were clustered in the periphery of the nucleus. Examples of mKOR-tdT are indicated by the yellow arrows. Scale bar: 20 μm.
Oxygen–glucose deprivation (OGD)-induced κ-opioid receptor (KOR) trafficking shared similar pattern of KOR agonist-induced KOR internalization. Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT) cells were untreated in control (A), incubated with 1 μM salvinorin A (SA), a selective KOR agonist, for 1 h (B), or treated with OGD for 1 h (C). Cells were fixed, mounted with 4',6-diamidino-2-phenylindole (DAPI), and observed by an inverted fluorescence microscope. SA induced KOR internalization, with a decrease of mKOR-tdT in the cell surface and an increase of crescent-shaped mKOR-tdT in the cytoplasm, which was similar to OGD-induced mKOR-tdT trafficking on the left. Merged images on the right show that both the internalized KORs by SA and OGD were clustered in the periphery of the nucleus. Examples of mKOR-tdT are indicated by the yellow arrows. Scale bar: 20 μm.
Fig. 4.
Oxygen–glucose deprivation (OGD)-induced κ-opioid receptor (KOR) trafficking shared similar pattern of KOR agonist-induced KOR internalization. Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT) cells were untreated in control (A), incubated with 1 μM salvinorin A (SA), a selective KOR agonist, for 1 h (B), or treated with OGD for 1 h (C). Cells were fixed, mounted with 4',6-diamidino-2-phenylindole (DAPI), and observed by an inverted fluorescence microscope. SA induced KOR internalization, with a decrease of mKOR-tdT in the cell surface and an increase of crescent-shaped mKOR-tdT in the cytoplasm, which was similar to OGD-induced mKOR-tdT trafficking on the left. Merged images on the right show that both the internalized KORs by SA and OGD were clustered in the periphery of the nucleus. Examples of mKOR-tdT are indicated by the yellow arrows. Scale bar: 20 μm.
×
Fig. 5.
κ-Opioid receptor (KOR) antagonist or internalization inhibitor blocked the oxygen–glucose deprivation (OGD)-induced KOR internalization. Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT) cells pretreated with 10 μM KOR-selective antagonist norbinaltorphimine or 30 µM dynamin 1 inhibitor Dyngo-4a for half an hour inhibited both salvinorin A (SA) and OGD-induced KOR internalization. Examples of mKOR-tdT are indicated by the yellow arrows. (A) SA or OGD, (B) norbinaltorphimine plus SA or OGD, (C) Dyngo-4a plus SA or OGD. Scale bar: 20 μm. (B) Cell viability by 3-(4,5-dimethylthia-2ol-2-yl)-2,5- diphenyl-tetrazolium bromide assay after 1 μM SA or OGD treatment of the N2A-mKOR-tdT cells for 1 h either with or without 10 μM norbinaltorphimine or 30 µM Dyngo-4a pretreatment for 30 min. Data are shown as mean ± SD (n = 6).
κ-Opioid receptor (KOR) antagonist or internalization inhibitor blocked the oxygen–glucose deprivation (OGD)-induced KOR internalization. Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT) cells pretreated with 10 μM KOR-selective antagonist norbinaltorphimine or 30 µM dynamin 1 inhibitor Dyngo-4a for half an hour inhibited both salvinorin A (SA) and OGD-induced KOR internalization. Examples of mKOR-tdT are indicated by the yellow arrows. (A) SA or OGD, (B) norbinaltorphimine plus SA or OGD, (C) Dyngo-4a plus SA or OGD. Scale bar: 20 μm. (B) Cell viability by 3-(4,5-dimethylthia-2ol-2-yl)-2,5- diphenyl-tetrazolium bromide assay after 1 μM SA or OGD treatment of the N2A-mKOR-tdT cells for 1 h either with or without 10 μM norbinaltorphimine or 30 µM Dyngo-4a pretreatment for 30 min. Data are shown as mean ± SD (n = 6).
Fig. 5.
κ-Opioid receptor (KOR) antagonist or internalization inhibitor blocked the oxygen–glucose deprivation (OGD)-induced KOR internalization. Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT) cells pretreated with 10 μM KOR-selective antagonist norbinaltorphimine or 30 µM dynamin 1 inhibitor Dyngo-4a for half an hour inhibited both salvinorin A (SA) and OGD-induced KOR internalization. Examples of mKOR-tdT are indicated by the yellow arrows. (A) SA or OGD, (B) norbinaltorphimine plus SA or OGD, (C) Dyngo-4a plus SA or OGD. Scale bar: 20 μm. (B) Cell viability by 3-(4,5-dimethylthia-2ol-2-yl)-2,5- diphenyl-tetrazolium bromide assay after 1 μM SA or OGD treatment of the N2A-mKOR-tdT cells for 1 h either with or without 10 μM norbinaltorphimine or 30 µM Dyngo-4a pretreatment for 30 min. Data are shown as mean ± SD (n = 6).
×
The results from fluorescence imaging flow cytometry show that mKOR-tdT of cells in normoxia had a higher circularity index than that in hypoxia (fig. 6A). Single-cell imaging illustrated that OGD-induced intense fluorescence gathered in the intracellular space, while norbinaltorphimine inhibited such morphologic changes (fig. 6B).
Fig. 6.
Oxygen–glucose deprivation (OGD) decreased the circularity feature and increased contrast of mouse κ-opioid receptor–tdTomato fusion protein (mKOR-tdT) with imaging flow cytometry observation. Cells in normoxia had a higher circularity index than that in hypoxia (A). OGD induced a decrease in circularity and an increase in the contrast of mKOR-tdT. κ-Opioid receptor antagonist norbinaltorphimine inhibited this change (B).
Oxygen–glucose deprivation (OGD) decreased the circularity feature and increased contrast of mouse κ-opioid receptor–tdTomato fusion protein (mKOR-tdT) with imaging flow cytometry observation. Cells in normoxia had a higher circularity index than that in hypoxia (A). OGD induced a decrease in circularity and an increase in the contrast of mKOR-tdT. κ-Opioid receptor antagonist norbinaltorphimine inhibited this change (B).
Fig. 6.
Oxygen–glucose deprivation (OGD) decreased the circularity feature and increased contrast of mouse κ-opioid receptor–tdTomato fusion protein (mKOR-tdT) with imaging flow cytometry observation. Cells in normoxia had a higher circularity index than that in hypoxia (A). OGD induced a decrease in circularity and an increase in the contrast of mKOR-tdT. κ-Opioid receptor antagonist norbinaltorphimine inhibited this change (B).
×
After 4 h of OGD, KOR trafficking could no longer be blocked or reversed by reoxygenation (data not shown) due to irreversible cellular damage induced by prolonged hypoxia. This was consistent with the results of the MTT assay. Treatment of cells with glibenclamide, iberiotoxin, or sulpiride failed to block the OGD-induced KOR internalization, indicating that adenosine triphosphate-sensitive and high-conductance calcium-activated potassium channels, as well as dopamine D2 receptors, are not involved in OGD-induced KOR internalization (data not shown).
OGD-induced Subcellular KOR Trafficking Observed by Electron Microscopy
Immunogold labeling of ultrathin sections of the cells revealed subcellular localization of KORs. Under normal oxygen, the cell was in good shape, and the nucleus was large-spindle (fig. 7A); however, under hypoxia, many swollen mitochondria were present, the nucleus was shrunken, and the nucleolus was observed all over the nucleus (fig. 7B). Under normal oxygen, most gold particles associated with KORs were distributed on the cell surface (fig. 7C). After 1 h of OGD treatment, gold particles moved into the cytoplasm and distributed in the periphery of the nucleus. Fewer particles were found on the cell surface (fig. 7D). Perinuclear distribution coincided with the results of the fluorescence microscopy. To quantify the movement of KORs, 10 sections with distinct nuclei were examined, and the gold particles were counted (5 sections on 1 grid, 2 grids under each condition). There was no significant difference in quantity of total particles between the control group and the OGD group. During normoxia, 62.7 and 37.3% particles were associated with the cell surface and the cytoplasm, respectively. OGD significantly increased the portion of gold particles to 85% in the cytoplasm (P < 0.01; fig. 7, E and F).
Fig. 7.
κ-Opioid receptor (KOR) trafficking under oxygen–glucose deprivation (OGD) was observed with immunoelectron microscopy. (A) Healthy cells in normoxia had a large spindle-shaped nucleus; (B) nucleus shrinkage and cytoplasmic vacuolization were observed in the OGD group; (C) KOR-conjugated gold particles located around the cell membrane (green arrows = cell membrane; blue arrows = gold particles); (D) KOR-conjugated gold particles distributed in the periphery of the nucleus area. (E) There was no difference in the total gold particles between the normoxia and OGD groups; however, more cytoplasmic particles were founded in the OGD group (n = 10 per group). (F) Distribution of gold particles within the cytoplasm after 1 h of normoxia (control) or OGD conditions (n = 10 per group). Data are shown as mean ± SD. *P < 0.001. CM = cell membrane particles number; CP = cytoplasmic particles number; Total = the total number of particles in one cell.
κ-Opioid receptor (KOR) trafficking under oxygen–glucose deprivation (OGD) was observed with immunoelectron microscopy. (A) Healthy cells in normoxia had a large spindle-shaped nucleus; (B) nucleus shrinkage and cytoplasmic vacuolization were observed in the OGD group; (C) KOR-conjugated gold particles located around the cell membrane (green arrows = cell membrane; blue arrows = gold particles); (D) KOR-conjugated gold particles distributed in the periphery of the nucleus area. (E) There was no difference in the total gold particles between the normoxia and OGD groups; however, more cytoplasmic particles were founded in the OGD group (n = 10 per group). (F) Distribution of gold particles within the cytoplasm after 1 h of normoxia (control) or OGD conditions (n = 10 per group). Data are shown as mean ± SD. *P < 0.001. CM = cell membrane particles number; CP = cytoplasmic particles number; Total = the total number of particles in one cell.
Fig. 7.
κ-Opioid receptor (KOR) trafficking under oxygen–glucose deprivation (OGD) was observed with immunoelectron microscopy. (A) Healthy cells in normoxia had a large spindle-shaped nucleus; (B) nucleus shrinkage and cytoplasmic vacuolization were observed in the OGD group; (C) KOR-conjugated gold particles located around the cell membrane (green arrows = cell membrane; blue arrows = gold particles); (D) KOR-conjugated gold particles distributed in the periphery of the nucleus area. (E) There was no difference in the total gold particles between the normoxia and OGD groups; however, more cytoplasmic particles were founded in the OGD group (n = 10 per group). (F) Distribution of gold particles within the cytoplasm after 1 h of normoxia (control) or OGD conditions (n = 10 per group). Data are shown as mean ± SD. *P < 0.001. CM = cell membrane particles number; CP = cytoplasmic particles number; Total = the total number of particles in one cell.
×
Discussion
In this study, the effect of OGD on the translocation of KOR in the mouse Neuro2A neuroblastoma cells was investigated. In normoxia, the majority of KORs were located on the cell membrane. OGD induced significant internalization of KORs. Such internalization can be blocked by specific KOR antagonists or reversed by reoxygenation. OGD-induced KOR internalization might share the similar mechanism with the agonist-activated KOR internalization, since specific KOR antagonists can block it. These findings suggest that KOR might play a critical role in modulating hypoxia.
GPCR-mediated signal transduction depends on the receptor density and activity for ligand binding on the cell surface.30,31  Agonist activation triggers changes in receptor density and activity by three distinct processes. Receptor desensitization (from seconds to hours) is the alternation of the receptor function but not receptor amount, while internalization (from minutes to hours) is a rapid agonist-induced receptor movement from cell surface to cytoplasm. Reversible movement is critical for receptor resensitization. Down-regulation (from hours to days) refers to a reduction of total receptor due to less gene expression.30–32  Previous study demonstrated that 30-min U50,488 exposure promoted 30 to 40% of total KOR internalization, which began with phosphorylation of KORs by GPCR kinases after β-arrestin recruitment and KOR uncoupling from G protein. Uncoupled KORs then clustered into the clathrin-coated pits to form endocytic vesicles. Removal of U50,488 resulted in gradual recycling of KORs to the cell surface for an hour.11,32  It was also reported that 15-min acute salt loading could elicit increased KORs in the presynaptic plasma membrane, which was due to cell membrane depolarization.32  All the above reports indicate that multiple external factors such as agonist treatment or physical stimulation might change KOR distribution.
Explicit KOR distribution is important for KOR-related signal transduction and therapeutic efficacy. Prolonged hypoxia (9%, 7 days) selectively decreased DOR ligand binding but not MOR or KOR in mice brains, and such changes might be due to the hypoxia-stimulated endogenous δ-agonist release and DOR down-regulation.33  KOR ligand binding decreased between 6 and 24 h after permanent occlusion of the distal middle cerebral artery in the infarct frontoparietal cortices of mice, which was slower than DOR ligand binding (1 to 3 h) and MOR ligand binding (6 to 12 h). Similarly, KOR ligand binding decreased later (12 to 48 h after occlusion of the distal middle cerebral artery) than DOR ligand binding (6 to 12 h) in the infarct border zone.34  The ligand binding affinity reflects both the available receptor expression and the receptor activity. However, the change in receptor distribution by this method is determined indirectly, and the subcellular distribution of opioid receptor cannot be revealed under hypoxia.
In this study, the change in KOR trafficking was observed directly. The redistribution time window and phenomenon are similar to agonist-induced KOR internalization, which can be blocked by the KOR antagonist. Endocytosis inhibitors can block such events. All these findings support the notion that OGD induces KOR-activated internalization in Neuro2A cells. External stress was reported to activate KOR via increased endogenous dynorphin release in vivo.35  However, the mechanisms involved in OGD-induced KOR activation and internalization were different since there should have been no endorphin secretion in an in vitro model. Exposure to severe hypoxia for 30 min leads to significant 5-hydroxytryptamine (serotonin) receptor internalization by up to 40% in the adult ovine common carotid artery, which might be due to GPCR kinase-related receptor phosphorylation.36  Similarly, 1-h OGD was found to decrease γ-aminobutyric acid receptor type A in cultured hippocampal neurons via accelerated internalization. Decreased adenosine triphosphate levels, increased levels of protein kinase C activity, and calcium-activated phosphatases might be involved in this process.37  Acute hypoxia first activates oxygen-regulated ion channels in the cell membrane, which triggers inhibition of the potassium channel, activation of the sodium channel, membrane depolarization, and potentiation of calcium influx.38–40  Mitogen-activated protein kinases (MAPKs), a family of serine–threonine protein kinases including extracellular signal-regulated kinase, c-Jun NH2-terminal kinase, and p38 proteins, are activated by diverse stimuli including cellular stress and hypoxia. Hypoxia induced phosphorylation of p38 and p42 or 44 MAPK in a neural-like PC12 cell line.41,42  In human ovarian carcinoma cells, activated p38 or MAPKs were significantly increased between 30 and 120 min after acute hypoxia and declined thereafter.43  Although it is not yet known which signal transduction cascades lead to hypoxic activated MAPKs, the increased calcium and mitochondrial reactive oxygen species might initiate phosphorylation of MAPKs.42,44,45  Similarly, stimulation of KOR was reported to activate MAPKs, which was independent of KOR internalization.14  It seems that both hypoxia and activation of KOR might have the same downstream cellular signaling pathway. Recently, it was reported that GPCR-mediated extracellular signal-regulated kinase activation had a reciprocal effect on GPCR trafficking, which reduced the cell-surface expression of GPCRs and increased the intracellular expression.46  Thus, it is unlikely that the acute or short-term hypoxia-induced KOR activation and internalization observed in this study share the similar mechanism noted above. Despite the effort to investigate the potential roles of some of the ion channels and dopamine receptors, we failed to elucidate the explicit molecular mechanism of hypoxia-induced KOR activation. Further studies are needed. Consistently, with long-time agonist-induced KOR down-regulation, exposure to OGD for more than 4 h impaired the KOR recycling. Neither reoxygenation nor antagonists could reverse such KOR movement with decreased KOR numbers in the cell membrane.
While we cannot apply the findings in this study to all the neuronal cells, we believe that the discovery related to KOR internalization using neuroblastoma cell is applicable to other neuronal cells since Salvinorin A, a highly selective KOR agonist, induced KOR internalization that can be blocked by KOR antagonist norbinaltorphimine. However, considering signal disruption activity of norbinaltorphimine,47  additional mechanistic studies are required to elucidate whether hypoxia may induce or disrupt KOR-dependent signaling pathways. The exact clinical implication and significance of the KOR internalization induced by hypoxia is not known based on the current in vitro data. It is possible that such KOR internalization is a protective physiologic mechanism to avoid harm from hypoxia for neurons. The indirect evidence for such statement is that Salvinorin A, the highly selective KOR agonist, can induce KOR internalization similar to that by hypoxia, and Salvinorin A could protect the brain from hypoxia and ischemia, resulting in improved neurologic outcome as we have demonstrated in a rodent model of brain ischemia.48  Further experiments are needed to investigate whether naïve KORs in primary neuronal cultures undergo similar trafficking as in neuroblastoma cells with KOR overexpression.
Conclusion
Hypoxia induces reversible KOR internalization, which was inhibited by selective KOR antagonists or dynamin inhibitor, and can be reversed by reoxygenation in neuroblastoma cells, indicating the modulating effects between KOR and hypoxia via KOR activation and dynamin-dependent mechanism.
Acknowledgments
The authors thank Lee-Yuan Liu-Chen, Ph.D., and Peng Huang, Ph.D., at the Center for Substance Abuse Research and Department of Pharmacology of Lewis Katz School of Medicine at Temple University, Philadelphia, Pennsylvania, for providing N2A-mKOR-tdT and N2A-mKOR-FLAG cells and substantial technical guidance and support related to these cells.
Research Support
Supported by grant Nos. K08-GM-093115 and 1R01GM111421 from the National Institutes of Health, Bethesda, Maryland (to Dr. Liu).
Competing Interests
The authors declare no competing interests.
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Fig. 1.
Oxygen–glucose deprivation (OGD) and cell viability. (A) All OGD groups of Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT) cells maintained with 95% N2 and 5% CO2 from 0.5 to 12 h had significantly decreased partial pressure of oxygen (mmHg) in culture medium compared with that in the control group without OGD (n = 6 per group). (B) There was no significant difference among partial pressure of carbon dioxide (mmHg) in culture medium with OGD of N2A-mKOR-tdT cells from 0.5 to 12 h (n = 6 per group). OGD-induced time-dependent decrease of cell viability in both (C) N2A-mKOR-tdT and (D) Neuro2A cells stably transfected with Flag-tagged mouse κ-opioid receptor (N2A-mKOR-FLAG) cells after 4 h of treatment (n = 12 per group). Data are shown as mean ± SD. *P < 0.001.
Oxygen–glucose deprivation (OGD) and cell viability. (A) All OGD groups of Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT) cells maintained with 95% N2 and 5% CO2 from 0.5 to 12 h had significantly decreased partial pressure of oxygen (mmHg) in culture medium compared with that in the control group without OGD (n = 6 per group). (B) There was no significant difference among partial pressure of carbon dioxide (mmHg) in culture medium with OGD of N2A-mKOR-tdT cells from 0.5 to 12 h (n = 6 per group). OGD-induced time-dependent decrease of cell viability in both (C) N2A-mKOR-tdT and (D) Neuro2A cells stably transfected with Flag-tagged mouse κ-opioid receptor (N2A-mKOR-FLAG) cells after 4 h of treatment (n = 12 per group). Data are shown as mean ± SD. *P < 0.001.
Fig. 1.
Oxygen–glucose deprivation (OGD) and cell viability. (A) All OGD groups of Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT) cells maintained with 95% N2 and 5% CO2 from 0.5 to 12 h had significantly decreased partial pressure of oxygen (mmHg) in culture medium compared with that in the control group without OGD (n = 6 per group). (B) There was no significant difference among partial pressure of carbon dioxide (mmHg) in culture medium with OGD of N2A-mKOR-tdT cells from 0.5 to 12 h (n = 6 per group). OGD-induced time-dependent decrease of cell viability in both (C) N2A-mKOR-tdT and (D) Neuro2A cells stably transfected with Flag-tagged mouse κ-opioid receptor (N2A-mKOR-FLAG) cells after 4 h of treatment (n = 12 per group). Data are shown as mean ± SD. *P < 0.001.
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Fig. 2.
Oxygen–glucose deprivation (OGD)-induced κ-opioid receptor (KOR) trafficking of Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT) and Flag-tagged mouse κ-opioid receptor (N2A-mKOR-FLAG) cells with a similar pattern. N2A-mKOR-tdT cells expressing mKOR–tdTomato fusion protein (left, red) were either untreated (A) or treated with OGD for 0.5 h (B) or 1 h (C) before the fixation. N2A-mKOR-FLAG cells labeled with anti-FLAG monoclonal antibody were fixed followed by immunostaining with anti-FLAG Alexa Fluor 488-conjugated antibody (right, green). Both mKOR-tdT and mKOR-FLAG were distributed on the cell surface of the untreated group. However, both mKOR-tdT and mKOR-FLAG were internalized as punctuated under OGD. Moreover, OGD-induced change in KOR trafficking could be reversed by reoxygenation (D). The figures represent one of the three independent experiments with similar results. Examples of mKOR-tdT and mKOR-FLAG are indicated by the yellow arrows. Scale bar: AD, 20 μm.
Oxygen–glucose deprivation (OGD)-induced κ-opioid receptor (KOR) trafficking of Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT) and Flag-tagged mouse κ-opioid receptor (N2A-mKOR-FLAG) cells with a similar pattern. N2A-mKOR-tdT cells expressing mKOR–tdTomato fusion protein (left, red) were either untreated (A) or treated with OGD for 0.5 h (B) or 1 h (C) before the fixation. N2A-mKOR-FLAG cells labeled with anti-FLAG monoclonal antibody were fixed followed by immunostaining with anti-FLAG Alexa Fluor 488-conjugated antibody (right, green). Both mKOR-tdT and mKOR-FLAG were distributed on the cell surface of the untreated group. However, both mKOR-tdT and mKOR-FLAG were internalized as punctuated under OGD. Moreover, OGD-induced change in KOR trafficking could be reversed by reoxygenation (D). The figures represent one of the three independent experiments with similar results. Examples of mKOR-tdT and mKOR-FLAG are indicated by the yellow arrows. Scale bar: A–D, 20 μm.
Fig. 2.
Oxygen–glucose deprivation (OGD)-induced κ-opioid receptor (KOR) trafficking of Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT) and Flag-tagged mouse κ-opioid receptor (N2A-mKOR-FLAG) cells with a similar pattern. N2A-mKOR-tdT cells expressing mKOR–tdTomato fusion protein (left, red) were either untreated (A) or treated with OGD for 0.5 h (B) or 1 h (C) before the fixation. N2A-mKOR-FLAG cells labeled with anti-FLAG monoclonal antibody were fixed followed by immunostaining with anti-FLAG Alexa Fluor 488-conjugated antibody (right, green). Both mKOR-tdT and mKOR-FLAG were distributed on the cell surface of the untreated group. However, both mKOR-tdT and mKOR-FLAG were internalized as punctuated under OGD. Moreover, OGD-induced change in KOR trafficking could be reversed by reoxygenation (D). The figures represent one of the three independent experiments with similar results. Examples of mKOR-tdT and mKOR-FLAG are indicated by the yellow arrows. Scale bar: AD, 20 μm.
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Fig. 3.
Oxygen–glucose deprivation (OGD)-induced κ-opioid receptor trafficking was further confirmed by confocal microscopy. Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT; left, red) and Flag-tagged mouse κ-opioid receptor (N2A-mKOR-FLAG) cells (right, green) were either untreated (A) or treated with OGD for 1 h (B) after the fixation and immunostaining as described in figure 2. A single 10-µm layer out of the z-stack is shown. Examples of mKOR-tdT and mKOR-FLAG are indicated by the yellow arrows. Scale bar: A, B, 20 μm.
Oxygen–glucose deprivation (OGD)-induced κ-opioid receptor trafficking was further confirmed by confocal microscopy. Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT; left, red) and Flag-tagged mouse κ-opioid receptor (N2A-mKOR-FLAG) cells (right, green) were either untreated (A) or treated with OGD for 1 h (B) after the fixation and immunostaining as described in figure 2. A single 10-µm layer out of the z-stack is shown. Examples of mKOR-tdT and mKOR-FLAG are indicated by the yellow arrows. Scale bar: A, B, 20 μm.
Fig. 3.
Oxygen–glucose deprivation (OGD)-induced κ-opioid receptor trafficking was further confirmed by confocal microscopy. Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT; left, red) and Flag-tagged mouse κ-opioid receptor (N2A-mKOR-FLAG) cells (right, green) were either untreated (A) or treated with OGD for 1 h (B) after the fixation and immunostaining as described in figure 2. A single 10-µm layer out of the z-stack is shown. Examples of mKOR-tdT and mKOR-FLAG are indicated by the yellow arrows. Scale bar: A, B, 20 μm.
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Fig. 4.
Oxygen–glucose deprivation (OGD)-induced κ-opioid receptor (KOR) trafficking shared similar pattern of KOR agonist-induced KOR internalization. Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT) cells were untreated in control (A), incubated with 1 μM salvinorin A (SA), a selective KOR agonist, for 1 h (B), or treated with OGD for 1 h (C). Cells were fixed, mounted with 4',6-diamidino-2-phenylindole (DAPI), and observed by an inverted fluorescence microscope. SA induced KOR internalization, with a decrease of mKOR-tdT in the cell surface and an increase of crescent-shaped mKOR-tdT in the cytoplasm, which was similar to OGD-induced mKOR-tdT trafficking on the left. Merged images on the right show that both the internalized KORs by SA and OGD were clustered in the periphery of the nucleus. Examples of mKOR-tdT are indicated by the yellow arrows. Scale bar: 20 μm.
Oxygen–glucose deprivation (OGD)-induced κ-opioid receptor (KOR) trafficking shared similar pattern of KOR agonist-induced KOR internalization. Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT) cells were untreated in control (A), incubated with 1 μM salvinorin A (SA), a selective KOR agonist, for 1 h (B), or treated with OGD for 1 h (C). Cells were fixed, mounted with 4',6-diamidino-2-phenylindole (DAPI), and observed by an inverted fluorescence microscope. SA induced KOR internalization, with a decrease of mKOR-tdT in the cell surface and an increase of crescent-shaped mKOR-tdT in the cytoplasm, which was similar to OGD-induced mKOR-tdT trafficking on the left. Merged images on the right show that both the internalized KORs by SA and OGD were clustered in the periphery of the nucleus. Examples of mKOR-tdT are indicated by the yellow arrows. Scale bar: 20 μm.
Fig. 4.
Oxygen–glucose deprivation (OGD)-induced κ-opioid receptor (KOR) trafficking shared similar pattern of KOR agonist-induced KOR internalization. Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT) cells were untreated in control (A), incubated with 1 μM salvinorin A (SA), a selective KOR agonist, for 1 h (B), or treated with OGD for 1 h (C). Cells were fixed, mounted with 4',6-diamidino-2-phenylindole (DAPI), and observed by an inverted fluorescence microscope. SA induced KOR internalization, with a decrease of mKOR-tdT in the cell surface and an increase of crescent-shaped mKOR-tdT in the cytoplasm, which was similar to OGD-induced mKOR-tdT trafficking on the left. Merged images on the right show that both the internalized KORs by SA and OGD were clustered in the periphery of the nucleus. Examples of mKOR-tdT are indicated by the yellow arrows. Scale bar: 20 μm.
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Fig. 5.
κ-Opioid receptor (KOR) antagonist or internalization inhibitor blocked the oxygen–glucose deprivation (OGD)-induced KOR internalization. Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT) cells pretreated with 10 μM KOR-selective antagonist norbinaltorphimine or 30 µM dynamin 1 inhibitor Dyngo-4a for half an hour inhibited both salvinorin A (SA) and OGD-induced KOR internalization. Examples of mKOR-tdT are indicated by the yellow arrows. (A) SA or OGD, (B) norbinaltorphimine plus SA or OGD, (C) Dyngo-4a plus SA or OGD. Scale bar: 20 μm. (B) Cell viability by 3-(4,5-dimethylthia-2ol-2-yl)-2,5- diphenyl-tetrazolium bromide assay after 1 μM SA or OGD treatment of the N2A-mKOR-tdT cells for 1 h either with or without 10 μM norbinaltorphimine or 30 µM Dyngo-4a pretreatment for 30 min. Data are shown as mean ± SD (n = 6).
κ-Opioid receptor (KOR) antagonist or internalization inhibitor blocked the oxygen–glucose deprivation (OGD)-induced KOR internalization. Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT) cells pretreated with 10 μM KOR-selective antagonist norbinaltorphimine or 30 µM dynamin 1 inhibitor Dyngo-4a for half an hour inhibited both salvinorin A (SA) and OGD-induced KOR internalization. Examples of mKOR-tdT are indicated by the yellow arrows. (A) SA or OGD, (B) norbinaltorphimine plus SA or OGD, (C) Dyngo-4a plus SA or OGD. Scale bar: 20 μm. (B) Cell viability by 3-(4,5-dimethylthia-2ol-2-yl)-2,5- diphenyl-tetrazolium bromide assay after 1 μM SA or OGD treatment of the N2A-mKOR-tdT cells for 1 h either with or without 10 μM norbinaltorphimine or 30 µM Dyngo-4a pretreatment for 30 min. Data are shown as mean ± SD (n = 6).
Fig. 5.
κ-Opioid receptor (KOR) antagonist or internalization inhibitor blocked the oxygen–glucose deprivation (OGD)-induced KOR internalization. Neuro2A cells stably transfected with mouse κ-opioid receptor–tdTomato fusion protein (N2A-mKOR-tdT) cells pretreated with 10 μM KOR-selective antagonist norbinaltorphimine or 30 µM dynamin 1 inhibitor Dyngo-4a for half an hour inhibited both salvinorin A (SA) and OGD-induced KOR internalization. Examples of mKOR-tdT are indicated by the yellow arrows. (A) SA or OGD, (B) norbinaltorphimine plus SA or OGD, (C) Dyngo-4a plus SA or OGD. Scale bar: 20 μm. (B) Cell viability by 3-(4,5-dimethylthia-2ol-2-yl)-2,5- diphenyl-tetrazolium bromide assay after 1 μM SA or OGD treatment of the N2A-mKOR-tdT cells for 1 h either with or without 10 μM norbinaltorphimine or 30 µM Dyngo-4a pretreatment for 30 min. Data are shown as mean ± SD (n = 6).
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Fig. 6.
Oxygen–glucose deprivation (OGD) decreased the circularity feature and increased contrast of mouse κ-opioid receptor–tdTomato fusion protein (mKOR-tdT) with imaging flow cytometry observation. Cells in normoxia had a higher circularity index than that in hypoxia (A). OGD induced a decrease in circularity and an increase in the contrast of mKOR-tdT. κ-Opioid receptor antagonist norbinaltorphimine inhibited this change (B).
Oxygen–glucose deprivation (OGD) decreased the circularity feature and increased contrast of mouse κ-opioid receptor–tdTomato fusion protein (mKOR-tdT) with imaging flow cytometry observation. Cells in normoxia had a higher circularity index than that in hypoxia (A). OGD induced a decrease in circularity and an increase in the contrast of mKOR-tdT. κ-Opioid receptor antagonist norbinaltorphimine inhibited this change (B).
Fig. 6.
Oxygen–glucose deprivation (OGD) decreased the circularity feature and increased contrast of mouse κ-opioid receptor–tdTomato fusion protein (mKOR-tdT) with imaging flow cytometry observation. Cells in normoxia had a higher circularity index than that in hypoxia (A). OGD induced a decrease in circularity and an increase in the contrast of mKOR-tdT. κ-Opioid receptor antagonist norbinaltorphimine inhibited this change (B).
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Fig. 7.
κ-Opioid receptor (KOR) trafficking under oxygen–glucose deprivation (OGD) was observed with immunoelectron microscopy. (A) Healthy cells in normoxia had a large spindle-shaped nucleus; (B) nucleus shrinkage and cytoplasmic vacuolization were observed in the OGD group; (C) KOR-conjugated gold particles located around the cell membrane (green arrows = cell membrane; blue arrows = gold particles); (D) KOR-conjugated gold particles distributed in the periphery of the nucleus area. (E) There was no difference in the total gold particles between the normoxia and OGD groups; however, more cytoplasmic particles were founded in the OGD group (n = 10 per group). (F) Distribution of gold particles within the cytoplasm after 1 h of normoxia (control) or OGD conditions (n = 10 per group). Data are shown as mean ± SD. *P < 0.001. CM = cell membrane particles number; CP = cytoplasmic particles number; Total = the total number of particles in one cell.
κ-Opioid receptor (KOR) trafficking under oxygen–glucose deprivation (OGD) was observed with immunoelectron microscopy. (A) Healthy cells in normoxia had a large spindle-shaped nucleus; (B) nucleus shrinkage and cytoplasmic vacuolization were observed in the OGD group; (C) KOR-conjugated gold particles located around the cell membrane (green arrows = cell membrane; blue arrows = gold particles); (D) KOR-conjugated gold particles distributed in the periphery of the nucleus area. (E) There was no difference in the total gold particles between the normoxia and OGD groups; however, more cytoplasmic particles were founded in the OGD group (n = 10 per group). (F) Distribution of gold particles within the cytoplasm after 1 h of normoxia (control) or OGD conditions (n = 10 per group). Data are shown as mean ± SD. *P < 0.001. CM = cell membrane particles number; CP = cytoplasmic particles number; Total = the total number of particles in one cell.
Fig. 7.
κ-Opioid receptor (KOR) trafficking under oxygen–glucose deprivation (OGD) was observed with immunoelectron microscopy. (A) Healthy cells in normoxia had a large spindle-shaped nucleus; (B) nucleus shrinkage and cytoplasmic vacuolization were observed in the OGD group; (C) KOR-conjugated gold particles located around the cell membrane (green arrows = cell membrane; blue arrows = gold particles); (D) KOR-conjugated gold particles distributed in the periphery of the nucleus area. (E) There was no difference in the total gold particles between the normoxia and OGD groups; however, more cytoplasmic particles were founded in the OGD group (n = 10 per group). (F) Distribution of gold particles within the cytoplasm after 1 h of normoxia (control) or OGD conditions (n = 10 per group). Data are shown as mean ± SD. *P < 0.001. CM = cell membrane particles number; CP = cytoplasmic particles number; Total = the total number of particles in one cell.
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