Research ArticlePain

5-oxoETE triggers nociception in constipation-predominant irritable bowel syndrome through MAS-related G protein–coupled receptor D

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Science Signaling  18 Dec 2018:
Vol. 11, Issue 561, eaal2171
DOI: 10.1126/scisignal.aal2171
  • Fig. 1 Quantification of PUFA metabolites in mucosa of patients with IBS.

    (A) Heat map of PUFA metabolites quantified by LC-MS/MS. Data are shown in a matrix format: Each row represents a single PUFA metabolite, and each column represents a subgroup of patients. Each color patch represents the normalized quantity of PUFA metabolites (row) in a subgroup of patients (column), with a continuum of quantity from bright green (lowest) to bright red (highest). The pattern and length of the branches in the dendrograms reflect the relatedness of the PUFA metabolites. The dashed red line is the dendrogram distance used to cluster PUFA metabolites. (B) 5-oxoETE quantified by LC-MS/MS in the mucosa of HCs (white circles) and patients with the indicated type of IBS (black circles). Data are expressed in picograms per milligram of protein and presented as means ± SEM of 10 to 20 biopsies per group. Statistical analysis was performed using Kruskal-Wallis analysis of variance (ANOVA) and subsequent Dunn’s post hoc test. ***P < 0.001 compared to the HC group.

  • Fig. 2 5-oxoETE induces somatic and visceral hypersensitivity in vivo.

    (A to E) Eight-week-old male C57BL/6 J mice were subcutaneously injected with either Hanks’ balanced salt solution (HBSS; white circles) or 5-oxoETE (black circles) into hind footpads. (A) Somatic pain was monitored using the von Frey test at the indicated times after injection with 10 μM 5-oxoETE or HBSS. Data are means ± SEM of three independent experiments with five mice per group. Error bars indicate SEM. (B) The von Frey test was performed 30 min after injection of the indicated concentrations of 5-oxoETE. Data are means ± SEM of two experiments with six mice per group. (C) Mouse paw tissue samples were stained with hematoxylin and eosin (H&E) 6 hours after the administration of HBSS or 100 μM 5-oxoETE as indicated. Images are representative of two experiments with five mice per group. (D) Visceromotor response (VMR) to increasing pressures of colorectal distension (CRD) before and 30 min after intracolonic administration of 10 μM 5-oxoETE (black bars) or vehicle (40% ethanol; white bars). Data are means ± SEM of two experiments with 10 mice per group and are relative to the baseline recorded before treatment. (E) Colon tissue samples stained with H&E from mice treated with 40% ethanol or 10 μM 5-oxoETE as indicated. Images are representative of two experiments with five mice per group. Statistical analysis was performed using Kruskal-Wallis ANOVA and subsequent Dunn’s post hoc test. **P < 0.01 and ***P < 0.001 compared to control mice.

  • Fig. 3 5-oxoETE induces lumbar splanchnic nerve firing.

    (A) Example of a teased-fiber recording showing the lumbar splanchnic (that is, colon innervating) nerve response to ring application (7 min) of 5-oxoETE in mouse serosal afferents. Arrows indicate application and removal of 5-oxoETE. Data are representative of eight experiments in which 5-oxoETE elicited nerve discharge above baseline from a total of 21 teased fibers isolated from seven mice. (B) Mean change in firing per second in serosal receptive fields in response to 5-oxoETE compared with the response to vehicle (Krebs buffer). Data are means ± SEM of eight teased-fiber recordings (N = 7 mice) for 5-oxoETE and five teased-fiber recordings (N = 5 mice) for vehicle. Statistical analysis was performed using a Mann-Whitney t test. **P < 0.01 compared to vehicle. (C) Proportion of responses in lumbar splanchnic afferents to application of 5-oxoETE (n, number of teased-fiber recordings; N, number of mice).

  • Fig. 4 5-oxoETE induces an increase in [Ca

    2+]i in sensory neurons through a GPCR. (A) Representative trace of Ca2+ flux experiments in sensory neurons incubated in the absence of extracellular Ca2+/Mg2+ and exposed to 50 μM 5-oxoETE or vehicle (HBSS). (B) Ca2+ flux measurements in mouse sensory neurons exposed to the indicated concentrations of 5-oxoETE (black circles) or to vehicle (HBSS; white circles). Data are means ± SEM of seven independent experiments with three wells per condition and 60 to 80 neurons per well. (C) Amplitude of [Ca2+]iF/F; left) in human sensory neurons and the percentage of responding neurons (right) exposed to the indicated concentrations of 5-oxoETE (black bars) or to vehicle (HBSS; white bar). Data are means ± SEM of three independent experiments with three wells per condition and 20 to 53 neurons per well. (D) Percentages of IB4+ and IB4 mouse sensory neurons that responded to 10 μM 5-oxoETE (black bars) or HBSS (white bars). Data are means ± SEM of three independent experiments with three wells per condition and 60 to 80 neurons per well. (E) Effects of 30-min incubation with 10 μM U73122 (PLC inhibitor) or overnight incubation with pertussis toxin (PTX; 250 ng/ml) on 5-oxoETE–induced Ca2+ mobilization in mouse sensory neurons. Data are means ± SEM of five independent experiments with three wells per condition and 60 to 80 neurons per well. Statistical analysis was performed using Kruskal-Wallis ANOVA and subsequent Dunn’s post hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to HBSS.

  • Fig. 5 Expression of Mrgprd in sensory neurons.

    (A) Expression of Mrgprd (red) and Trpv1 (blue) mRNA transcripts as detected by single-cell qRT-PCR analysis (middle) of retrogradely labeled mouse colonic sensory neurons (left). Pie chart representation (right) of the expression (dark color) or not (light color) of Mrgprd and Trpv1 mRNA in FB+ neurons. Each segment represents a single colonic sensory neuron. (B) Representative images of GFP (green), CGRP immunoreactivity (red), and FB labeling (blue) in a T13 DRG from an MrgprdEGFP mouse in which FB was injected into the colon. Scale bar, 50 μm. Inset images are magnifications of the boxed areas in the largest images. (C and D) Expression of Mrgprd by immunostaining in a whole human T11 DRG (C) and in a primary culture of human sensory neurons using confocal microscopy (D). Pie chart representation of the immunoreactivity (dark color) or not (light color) of Mrgprd in Pgp9.5+ neurons. Each segment represents a single sensory neuron. Scale bars, 10 μm. Images are representative of two experiments with 10 slides per experiment (C) and of five experiments with two wells per experiment (D).

  • Fig. 6 Mrgprd expression is required for the intracellular Ca

    2+ mobilization and hypersensitivity induced by 5-oxoETE. (A) Left: Representative image of sensory neurons transfected with shRNA (red) and containing the Ca2+ indicator Fluo-4 AM (green). Right: Percentage of sensory neurons expressing control shRNA or Mrgprd-specific shRNA that responded to HBSS or 10 μM 5-oxoETE. Data are means ± SEM of six independent experiments with three wells per condition and 10 to 32 analyzed neurons per well. (B) Percentage of responding neurons (left) and amplitude of intracellular Ca2+ mobilization (ΔF/F; right) in mouse sensory neurons from Mrgprd-deficient mice exposed to vehicle (HBSS; white bar), 10 μM 5-oxoETE (black bar), or a mixture of GPCR agonists (GPCR mix: bradykinin, serotonin, and histamine, 10 μM each; gray bar). Data are means ± SEM of four independent experiments with three wells per condition and 20 to 50 neurons per well. (C) Effects of the indicated concentrations of 5-oxoETE and of 1 mM β-alanine (positive control) on the amplitude (ΔF/F) of Ca2+ mobilization in human embryonic kidney (HEK) cells transiently transfected with plasmid expressing Mrgprd or with an empty vector as a control. Data are means ± SEM of eight independent experiments with three wells per condition. (D) VMR in Mrgprd-deficient mice in response to increasing pressures of CRD before (baseline; white circles) and 30 min after intracolonic administration of 10 μM 5-oxoETE (black circles). Data are means ± SEM of two experiments of seven mice per experiment. Statistical analysis was performed using Kruskal-Wallis ANOVA and subsequent Dunn’s post hoc test. In (A), **P < 0.01 compared to the control shRNA/HBSS group; in (B), **P < 0.01 compared to the HBSS group; in (C), *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the corresponding CHO empty vector group.

  • Table 1 Percentage of FB+ neurons expressing Mrgprd, CGRP, or both in T13 DRGs per MrgprdEGFP mouse.
    AnimalMrgprd+CGRP+Mrgprd+and
    CGRP+
    A9 of 75 (12.0%)54 of 75 (72.0%)0 of 75 (0.0%)
    B2 of 38 (5.3%)24 of 38 (63.2%)0 of 38 (0.0%)
    C10 of 101 (9.9%)64 of 101 (63.4%)1 of 101 (1.0%)
    D1 of 60 (1.7%)46 of 60 (76.7%)0 of 60 (0.0%)
    Total (mean ± SD)7.2 ± 4.6%68.8 ± 6.7%0.3 ± 0.5%
  • Table 2 Characteristics of patients from which biopsies were collected for the quantification of PUFA metabolites.

    ControlIBS
    Number1450
    Age49 (20–76)43 (20–72)
    Sex ratio (F/M)8/632/18
    Bowel movements
      Diarrhea020
      Constipation020
      Mix010

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/561/eaal2171/DC1

    Fig. S1. Concentrations of PUFA metabolites in biopsies from patients with IBS.

    Fig. S2. Concentrations of PUFA metabolites in biopsies of all patients with IBS.

    Fig. S3. 5-oxoETE does not induce somatic or visceral inflammation in vivo.

    Fig. S4. 5-oxoETE induces Ca2+ flux in mouse sensory neurons.

    Fig. S5. Mrgprd immunoreactivity is observed in mouse colon.

    Fig. S6. Mrgprd immunoreactivity is not observed in the colon of Mrgprd-deficient mice.

  • This PDF file includes:

    • Fig. S1. Concentrations of PUFA metabolites in biopsies from patients with IBS.
    • Fig. S2. Concentrations of PUFA metabolites in biopsies of all patients with IBS.
    • Fig. S3. 5-oxoETE does not induce somatic or visceral inflammation in vivo.
    • Fig. S4. 5-oxoETE induces Ca2+ flux in mouse sensory neurons.
    • Fig. S5. Mrgprd immunoreactivity is observed in mouse colon.
    • Fig. S6. Mrgprd immunoreactivity is not observed in the colon of Mrgprd-deficient mice.

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