Research ArticlePain

TRPV1 promotes opioid analgesia during inflammation

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Science Signaling  02 Apr 2019:
Vol. 12, Issue 575, eaav0711
DOI: 10.1126/scisignal.aav0711
  • Fig. 1 Activation of TRPV1 mediates translocation of β-arrestin2 to the nucleus.

    (A to C) Spinning disk confocal imaging (A and B) and quantification (C) of β-arrestin2 nuclear translocation in HEK cells transfected with plasmids expressing TRPV1-mCherry (red; B) and β-arrestin2–YFP (green) and treated with either bath-applied 10 nM RTX at 37°C for the indicated times (A) or 1 μM capsaicin for 15 min (B). Arrows (A) indicate the nuclear localization of β-arrestin2–YFP. Scale bars, 10 μm. Data (C) are means ± SEM of at least 100 cells per condition from five experiments. **P < 0.001 and ***P < 0.0001 compared to time zero by an unpaired t test. (D) Illustration of the bystander BRET assay used to monitor the trafficking of β-arrestin2 to the nucleus. PM, plasma membrane; NM, nuclear membrane. (E and F) Time course of net bystander (E) and maximum (F) BRET measured in HEK cells expressing TRPV1, β2ARR-rLucII, and Renilla GFP-NLS and exposed to various concentrations of capsaicin (caps.) injected at time zero. Inset: Image of nuclear localization of the GFP-NLS signal in cells costained with 4′,6-diamidino-2-phenylindole (DAPI). In (F), data were normalized to control (ctrl; vehicle) conditions and are means ± SEM of three experiments. *P < 0.05 and **P < 0.001 by one-way analysis of variance (ANOVA) and Bonferroni post hoc test. (G and H) Western blotting and analysis of phosphorylated and total ERK in HEK cells transfected with plasmid expressing TRPV1 and treated with 10 nM RTX for the indicated times. Blots are representative of three experiments; data are means ± SEM of three experiments. (I and J) Effect of the Ca2+-chelator EGTA (10 mM) on RTX-induced pERK1/2 at 5 min as assessed by Western blotting (I) and on RTX-induced β-arrestin2 nuclear translocation at 15 min as assessed by confocal microscopy (J). Scale bar, 20 μm. Blots and image are representative of three experiments; data are means ± SEM of three experiments. *P < 0.05; **P < 0.01 by unpaired t test.

  • Fig. 2 Activation of TRPV1 prevents the DAMGO-induced interaction between MOR and β-arrestin2.

    (A and B) BRET measured over time in HEK cells expressing MOR-Rluc8 and β-arrestin2–Venus (βARR2-Venus) and exposed to 10 μM DAMGO (blue), 500 nM capsaicin (red), or both (green) injected a time zero, either in the absence (A) or presence (B) of TRPV1 expression. Data are means ± SEM of three experiments. (C) Quantification of the BRET signal, obtained from the experiments represented in (A) and (B). Data are means ± SEM of the last four points on the time-course curve, from three experiments. ***P < 0.001. (D) Dose-response curve of the net BRET between MOR-Rluc8 and β-arrestin2–Venus in the cells described in (B) exposed to increasing concentration of DAMGO (blue) or DAMGO and capsaicin (green). (E) Co-immunoprecipitation (IP) from HEK cells expressing MOR-YFP alone or with TRPV1-hemagglutinin (HA) and exposed to vehicle (lanes 1 to 3) or 1 μM DAMGO (lane 4). Blot is representative of three experiments. WB, Western blot. (F) Net BRET saturation curves between MOR-Rluc8 and TRPV1-YFP or TRPA1-YFP in HEK cells. Data are representative of three independent experiments.

  • Fig. 3 Activation of TRPV1 prevents MOR internalization.

    (A and B) Confocal microscopy of HEK cells expressing MOR-YFP (green) and TRPV1-mCherry (red) exposed to vehicle, 10 μM DAMGO, or 10 μM DAMGO and 1 μM capsaicin for 30 min. Scale bar, 10 μm. Number of MOR+ vesicles per cell (white arrows) were counted with ImageJ software. Data are means ± SEM of at least 100 cells per condition from three independent experiments. *P < 0.05 and ***P < 0.001 by Kruskal-Wallis test followed by Dunn’s post hoc test. ns, not significant. (C) Illustration of the bystander BRET-based approach for monitoring MOR trafficking to FYVE-tethered early endosomes (EE) in response to DAMGO or DAMGO and capsaicin. Upon treatment with DAMGO, the internalized MOR-rLuc8 induces an increase in BRET signal compared to unstimulated condition (left). On the basis our observations in (A), cotreatment with DAMGO and capsaicin (right) will reduce the BRET response by preventing receptor trafficking to FYVE-tethered early endosomes. (D) Bystander net BRET over time in HEK cells exposed to vehicle [phosphate-buffered saline (PBS)] or 10 μM DAMGO at 37°C for 20 min. Data were normalized to their respective baseline and are the means ± SEM from three experiments. (E) Bar graph representing the area under the curve (AUC) of the net bBRET data presented in (D). ***P < 0.01 by unpaired t test.

  • Fig. 4 Activation of TRPV1 prevents acute desensitization of DAMGO-mediated inhibition of the Cav2.2 current.

    (A) Representative Ca2+ current traces (evoked at +10 mV from a holding potential of −80 mV during 30 ms) in HEK cells expressing Cav2.2 (+α2δ1 and β2a), MOR1, and TRPV1 and acutely treated with vehicle (black symbol) or 1 μM DAMGO (blue symbol). The current was recorded in control conditions (left traces), after 1 hour of preincubation with 1 μM DAMGO (middle traces, acute desensitization), and 1 hour after preincubation with 1 μM DAMGO and 100 nM capsaicin (right traces). Experiments were repeated using β-arrestin2 knockout (ARRB2−/−) HEK cells with or without transfection with β-arrestin2 expression plasmid. Traces are representative of three independent experiments. (B) Percentage of peak current inhibition induced by 1 μM DAMGO in untreated (white), DAMGO-pretreated (blue), or DAMGO and capsaicin–pretreated cells (green) at 37°C. Data are means ± SEM from three independent experiments, from (left to right) 15, 9, 9, 10, 9, 10, 9, 10, and 10 HEK cells. *P < 0.01 by one-way ANOVA and Bonferroni post hoc test.

  • Fig. 5 Activation of TRPV1 promotes β-arrestin2 nuclear translocation, which results in reduced MOR desensitization in DRG neurons.

    (A) Representative confocal microscopy images of β-arrestin2 immunostaining (red) in TRPV1-expressing DRG neurons (green) isolated from Ai32/TRPV1-cre mice. Neurons were untreated (control) or stimulated with 100 nM capsaicin for 10 min. Images are representative of five independent experiments. (B) Linescan corresponding to the image in (A) and representing β-arrestin2 cellular distribution. a.u., arbitrary units. (C) Representative N-type current traces (recorded in the presence of 10 μM nifedipine) from a capsaicin-sensitive DRG neuron upon application of vehicle (black symbol) or 1 μM DAMGO (blue symbol). Recordings were done in DRG neurons from WT and TRPV1−/− mice for different conditions of pretreatment (vehicle, DAMGO, and DAMGO and capsaicin at 37°C) as also described in Fig. 4A. Inset: TRPV1 current elicited by 100 nM capsaicin at a holding potential of −80 mV. Data are representative of three independent experiments. (D) Percentage of peak current inhibition recorded in the experiment represented in (C), treated as indicated. Data are means ± SEM from three independent experiments, from (left to right) 8, 7, 8, 8, 6, and 15 DRG neurons. *P < 0.01 by one-way ANOVA and Bonferroni post hoc test.

  • Fig. 6 Peripheral endogenous opioidergic control of inflammatory pain is absent in TRPV1-deficient mice.

    (A) Mechanical sensitivity in mice assessed by PWTs using a dynamic plantar aesthesiometer. CFA was injected into the plantar surface of the right hind paw of WT (left graph) or TRPV1−/− (right) male mice. Mice received daily injections of 10 μl of either PBS (blue circles; 10 mice per group) or Nal-M (Nal-Meth; 2 mg/ml; red squares) (10 WT and 8 TRPV1−/− mice per group) in the ipsilateral ankle from days 4 to 14, 30 min before assessment of mechanical threshold. Data are means ± SEM of PWT measured in the inflamed (CFA) ipsilateral paw (color) and saline-injected contralateral paw (gray) normalized to baseline. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-way ANOVA and Bonferroni post hoc test. (B) Area under the PWT curves in (A) between days 6 and 10 were calculated and presented as the percentage of PWT normalized to vehicle-treated animals for each genotype. Data are means, min-max. ***P < 0.001 (or not significant) by Mann-Whitney U test.

  • Fig. 7 TRPV1 prevents peripheral opioid desensitization.

    (A) Schematic illustration of the drug administration paradigm. Mice (WT or TRPV1−/−) were treated with CFA at day 0 (D0) and received two daily injections of a morphine or saline solution (10 mg/kg). On day 3, potency of peripheral opioid–mediated analgesia was evaluated by measuring antinociceptive action of escalating doses of central (intrathecal) or local (paw) injection of morphine. (B) Dose-response curve of intrathecal morphine in WT (left) and TRPV1−/− (right) mice treated for 3 days after CFA injection with saline (CFA-SA; blue line) or morphine solution (MS; red line). (C) ED50 values as measured on day 3 after CFA treatment, from (B) (WT mice, n = 8; TRPV1−/− mice, n = 5). (D and E) As in (B) and (C), except for peripheral (paw) injection of morphine solution. (E: WT-CFA-Saline, n = 7; WT-CFA-MS, n = 5/TRPV1-CFA-Saline, n = 5; TRPV1-CFA-MS, n = 6). Data in (B) to (E) are means ± SD; each symbol represents one mouse. **P < 0.01 by regular two-way ANOVA and Sidak’s post hoc test.

  • Fig. 8 Model of the TRPV1-MOR interplay during opioid desensitization.

    Activation of MOR by exogenous or endogenous opioids mediates antinociception through the inhibition of N-type VGCCs in DRG neurons. Receptor desensitization and internalization is mediated by cellular signaling cascades, including the GRK-mediated phosphorylation of MOR, which in turn elicits β-arrestin2–dependent internalization of the receptor into early endosomes. Our data suggest that stimulation of TRPV1 with capsaicin or inflammatory mediators (protons, lipids, and temperature) promotes the translocation of β-arrestin2 to the nucleus and thus prevents MOR desensitization and internalization, facilitating inhibition of N-type channels.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/12/575/eaav0711/DC1

    Fig. S1. The translocation of β-arrestin2 to the nucleus is dependent on TRPV1 activation and accompanied by a transient PKCβII-dependent phosphorylation of ERK1/2.

    Fig. S2. Activation of TRPV1 prevents MOR and PAR2 from interacting with β-arrestin2.

    Fig. S3. The nuclear translocation of β-arrestin2 after TRPV1 stimulation is associated with impaired MOR internalization.

    Fig. S4. DAMGO and capsaicin have no detectable effects on CaV2.2 inhibition in the absence of MOR and TRPV1.

    Fig. S5. Peripheral endogenous control of inflammatory pain is absent in female TRPV1-deficient mice.

    Fig. S6. Paw edema, T lymphocyte infiltration, and opioid production are not altered in TRPV1−/− mice.

  • This PDF file includes:

    • Fig. S1. The translocation of β-arrestin2 to the nucleus is dependent on TRPV1 activation and accompanied by a transient PKCβII-dependent phosphorylation of ERK1/2.
    • Fig. S2. Activation of TRPV1 prevents MOR and PAR2 from interacting with β-arrestin2.
    • Fig. S3. The nuclear translocation of β-arrestin2 after TRPV1 stimulation is associated with impaired MOR internalization.
    • Fig. S4. DAMGO and capsaicin have no detectable effects on CaV2.2 inhibition in the absence of MOR and TRPV1.
    • Fig. S5. Peripheral endogenous control of inflammatory pain is absent in female TRPV1-deficient mice.
    • Fig. S6. Paw edema, T lymphocyte infiltration, and opioid production are not altered in TRPV1−/− mice.

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