Cutaneous nociception evoked by 15-delta PGJ2 via activation of ion channel TRPA1

Abstract

Background: A number of prostaglandins (PGs) sensitize dorsal root ganglion (DRG) neurons and contribute to inflammatory hyperalgesia by signaling through specific G protein-coupled receptors (GPCRs). One mechanism whereby PGs sensitize these neurons is through modulation of "thermoTRPs," a subset of ion channels activated by temperature belonging to the Transient Receptor Potential ion channel superfamily. Acrid, electrophilic chemicals including cinnamaldehyde (CA) and allyl isothiocyanate (AITC), derivatives of cinnamon and mustard oil respectively, activate thermoTRP member TRPA1 via direct modification of channel cysteine residues.

Results: Our search for endogenous chemical activators utilizing a bioactive lipid library screen identified a cyclopentane PGD2 metabolite, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), as a TRPA1 agonist. Similar to CA and AITC, this electrophilic molecule is known to modify cysteines of cellular target proteins. Electophysiological recordings verified that 15d-PGJ2 specifically activates TRPA1 and not TRPV1 or TRPM8 (thermoTRPs also enriched in DRG). Accordingly, we identified a population of mouse DRG neurons responsive to 15d-PGJ2 and AITC that is absent in cultures derived from TRPA1 knockout mice. The irritant molecules that activate TRPA1 evoke nociceptive responses. However, 15d-PGJ2 has not been correlated with painful sensations; rather, it is considered to mediate anti-inflammatory processes via binding to the nuclear peroxisome proliferator-activated receptor gamma (PPARgamma). Our in vivo studies revealed that 15d-PGJ2 induced acute nociceptive responses when administered cutaneously. Moreover, mice deficient in the TRPA1 channel failed to exhibit such behaviors.

Conclusion: In conclusion, we show that 15d-PGJ2 induces acute nociception when administered cutaneously and does so via a TRPA1-specific mechanism.

Figure 1

FLIPR-based library screen of 15d-PGJ2 activation of TRPA1. A. Fluorescence increases of TRPA1-expressing or mock transfected controls in response to a 50 μM 15d-PGJ2 stimulus. Traces represent average increases of cells from a 96-well plate. B. Fluorescence increases shown as minigraphs within 48 wells of TRPA1-expressing cells in response to increasing concentrations of 15d-PGJ2. All concentrations are in μM. 100 μM ATP and buffer were delivered as controls (the strain of CHO cell used exhibits endogenous ATP response and was used to ensure cell viability after compound addition).

Figure 2

15d-PGJ₂ activates TRPA1, but not TRPV1 or TRPM8, expressed in HeLa cells. A. Whole-cell current was recorded at a membrane potential of -40 mV (n = 3). B. A cell-attached patch shows activation of TRPA1 by 15d-PGJ₂. Pipette potential was held at -60 mV to record outward current (n = 4). 15d-PGJ₂ increased the channel activity (NPo) from a basal level of 0.03 ± 0.01 to 2.10 ± 0.30. C. A cell-attached patch shows activation of TRPA1 by 15d-PGJ₂ and AITC. Pipette potential was held at +40 mV to record inward current. The bars in the graph represent the mean ± SD of 4 determinations, and are significantly different from each other. The channel activity (NPo) elicited by AITC was 6.6 ± 1.4. D-E. In HeLa cells expressing TRPV1 or TRPM8, 15d-PGJ₂ had no effect, whereas capsaicin or menthol respectively, activated TRPV1 or TRPM8 (n = 4). Pipette potential was held at -40 mV. The large activation for TRPV1 by capsaicin could not be recorded at the amplifier gain setting used to show the lack of effect of 15d-PGJ2, but full washout was always observed following removal of capsaicin.

Figure 3

PGD₂ and its metabolites do not activate TRPA1. A-C. Cell-attached patches show lack of activation of TRPA1 by 12d-PGJ₂, PGJ₂ and PGD2. In the same cells, AITC caused strong activation (n = 5 each). Pipette potential was held at +40 mV.

Figure 4

15d-PGJ₂ specifically activates TRPA1-expressing DRG neurons. Traces represent increased fluorescence ratios of DRG neurons cultured from TRPA1 WT (A) or knockout (B) mice in response to 15d-PGJ₂, AITC and capsaicin. Panels A and B illustrate responses of neurons from separate calcium experiments (~100 neurons per experiment are illustrated). All experiments were performed in quadruplicate from cultures derived from 10 animals per genotype. Compound application is indicated by black bars (Caps, Capsaicin).

Figure 5

Intraplantar injection of 15d-PGJ₂ causes acute nociceptive responses via TRPA1. (A) 10 μL of vehicle (10% DMSO in saline) or 15 nmol 15d-PGJ₂ was injected into the hindpaw of C57BL/6J mice (n = 5 per group) and nociceptive behaviors (licking and lifting of the paw) for 10 minutes. 15d-PGJ₂ caused significant nociceptive responses compared to vehicle. (B) 15d-PGJ₂-induced nociceptive behaviors are absent in TRPA1 knockout mice (n = 5 per group). ***p < 0.001