Proteinase-activated receptor 2-mediated potentiation of transient receptor potential vanilloid subfamily 1 activity reveals a mechanism for proteinase-induced inflammatory pain

Abstract

Proteinase-activated receptor (PAR) 2 is expressed on a subset of primary afferent neurons and involved in inflammatory nociception. Transient receptor potential vanilloid subfamily 1 (TRPV1) is a sensory neuron-specific cation channel that responds to capsaicin, protons, or heat stimulus. Here, we show that TRPV1 is coexpressed with PAR2 but not with PAR1 or PAR3, and that TRPV1 can functionally interact with PAR2. In human embryonic kidney 293 cells expressing TRPV1 and PAR2, PAR2 agonists increased capsaicin- or proton-evoked TRPV1 currents through a PKC-dependent pathway. After application of PAR2 agonists, temperature threshold for TRPV1 activation was reduced from 42 degrees C to well below the body temperature. PAR2-mediated Fos expression in spinal cord was decreased in TRPV1-deficient mice. The functional interaction was also observed in mouse DRG neurons and proved at a behavioral level. These represent a novel mechanism through which trypsin or tryptase released in response to tissue inflammation might trigger the sensation of pain by PAR2 activation.

Figure 1.

PAR2, but not PAR1 and PAR3, coexpressed with TRPV1 in rat DRG neurons. A–C, Microphotographs show localization of PAR2 with TRPV1 by immunofluorescence. A, Detection of PAR2-LI (red) in many small-sized and some medium- to large-sized neurons. B, TRPV1-LI (green) in small-sized neurons. C, Colocalization with TRPV1 and PAR2 in DRG neurons. Merged, Yellow. Arrowheads represent neurons expressing both TRPV1 and PAR2, and arrows represent neurons expressing PAR2 but not TRPV1. D, E, Colocalization of TRPV1 protein with PAR1 (D) or PAR3 (E) mRNA. In situ hybridization histochemistry of PARs, using ³⁵S-labeled antisense probes and visualized as clusters of silver grains, was combined with immunohistochemistry of TRPV1 by using DAB staining method. Arrowheads represent neurons expressing TRPV1 but not PARs, and arrows represent neurons expressing PARs but not TRPV1. F, Percentage of neurons expressing PARs in TRPV1-positive ones. Scale bars, 50 μm.

Figure 2.

The PAR2 agonist SL-NH₂ potentiates or sensitizes capsaicin- and proton-activated currents in a PKC-dependent manner in HEK293 cells. A, A representative trace of increase of capsaicin (Cap, 20 nm)-activated current in transfected HEK293 cells expressing rat TRPV1 and rat PAR2. Cells were perfused for 2 min with solution containing SL-NH₂ (100 μm) before capsaicin applications. Capsaicin was reapplied 0.5, 3, and 7 min after SL-NH₂ treatment. Holding potential (V_h)= –60 mV. B, Time course of the potentiating effect after SL-NH₂ treatment. Numbers in parentheses indicate cells tested. *p < 0.05 versus before SL-NH₂ treatment (Bef); unpaired t test. C, A PKC-dependent pathway is involved in the SL-NH₂-induced potentiation of capsaicin-activated currents. Capsaicin was reapplied 2 min after exposure to bath solution with or without SL-NH₂(SL–) or LR-NH₂(LR–). Currents were normalized to values first induced by capsaicin application in the absence of SL-NH₂ or LR-NH₂. In some experiments, calphostin C (Calp.C) at 1 μ m or PKCϵ translocation inhibitor (PKCϵ-I) at 200 μm was included in the pipette solution. Cont, Control group (preperfused with bath solution without SL-NH₂ before reapplication of capsaicin); S502A/S800, cells expressing a TRPV1 mutant lacking sites for PKC-dependent phosphorylation. Numbers in parentheses indicate cells tested. *p < 0.05 versus Cont;^ζ p < 0.05 versus LR-NH₂; ^#p < 0.05 versus SL-NH₂; unpaired t test. D, Capsaicin dose–response curves for TRPV1 activation in the absence (•) and presence (○) of 100 μm SL-NH₂. Currents were normalized to the currents maximally activated by 1 μm capsaicin in the absence of SL-NH₂. The figure shows averaged data fitted with the Hill equation. EC₅₀ = 153 nm and Hill coefficient = 1.96 in the absence of SL-NH₂. EC₅₀ = 100 nm and Hill coefficient = 1.76 in the presence of SL-NH₂. Values were obtained from 32 different cells. E, A representative trace of increase of proton (pH 6.2)-activated current in transfected HEK293 cells expressing TRPV1 and PAR2. F, SL-NH₂ but not LR-NH₂ potentiates proton-evoked TRPV1 responses. V_h= –60 mV. Currents were normalized to values induced by first proton application in the absence of SL-NH₂ or LR-NH₂. Numbers in parentheses indicate cells tested. *p < 0.05 versus Cont; ^ζ p < 0.05 versus LR-NH₂; unpaired t test.

Figure 3.

Temperature threshold for TRPV1 activation was reduced in the presence of SL-NH₂ in HEK293 cells. A, B, Representative temperature-response profiles of heat-activated currents in the absence (A) and presence (B) of SL-NH₂ (100 μm). Dashed lines show the threshold temperature for heat activation of TRPV1. V_h = –60 mV. C, Temperature thresholds for TRPV1 activation in the presence of SL-NH₂ (black bar) (30.8±2.5°C;n=12) and 10 min after SL-NH₂ treatment (hatched bar) (32.0 ± 3.8°C; n = 7) were significantly lower than that in the absence of SL-NH₂ (41.6 ± 0.5°C; n = 15). Temperature ramp was applied 0.5, 3, 7, and 10 min after SL-NH₂ treatment. Numbers in parentheses indicate cells tested. *p < 0.05 versus SL-NH₂ (–); unpaired t test. Threshold was defined as a temperature at which clear current increase was observed in the temperature-response profile.

Figure 4.

PAR2 agonists potentiate capsaicin-activated currents in mouse DRG neurons. A–C, Representative traces of increase of the capsaicin (Cap, 100 nm)-activated currents by SL-NH₂ (100 μm)(A), tryptase (100 nm)(B), or trypsin (50 nm)(C). V_h = –60 mV. D, Effects of PAR2 agonists and PKC inhibitors on the capsaicin-activated currents. Currents were normalized to the currents evoked initially by capsaicin in the absence of the additives. Capsaicin was reapplied 2 min after exposure to these solutions. In some experiments, calphostin C (Calp. C) at 1 μm or PKCϵ translocation inhibitor (PKCϵ-I) at 200 μm was included in the pipette solution. Cont, Control group (preperfused with bath solution without any additives before reapplication of capsaicin); LR–, LR-NH₂;SL–, SL-NH₂. Numbers in parentheses indicate cells tested. *p < 0.05; **p < 0.01 versus Cont.;^ζ p < 0.05 versus LR-NH₂; ^#p < 0.05 versus SL-NH₂;^ψ p < 0.05 versus trypsin; unpaired t test.

Figure 5.

SL-NH₂-induced thermal hyperalgesia and mechanical allodynia were attenuated in TRPV1-deficient mice. A, B, Reduction of paw withdrawal latency to radiant lamp (A) and 50% paw withdrawal threshold to von Frey filaments (B) by intraplantar injection of SL-NH₂(10 μg per paw) was significantly attenuated in TRPV1-deficient mice (KO-SL). *p < 0.05 versus wild-type mice treated with SL-NH₂ (WT-SL); two-way repeated ANOVA followed by Fisher's PLSD; ^#p < 0.05 versus WT-SL; unpaired t test; ^† p < 0.05 versus wild-type mice treated with LR-NH₂ (10 μg per paw) (WT-LR); two-way repeated ANOVA followed by Fisher's PLSD. ^ψ p < 0.05; ^ψψ p < 0.001; ^ψψψ p < 0.0001 versus WT-SL; unpaired t test; n = 6 per group.

Figure 6.

PAR2 agonist-induced Fos expression in dorsal horn is significantly reduced in TRPV1-deficient mice. A–C, Microphotographs show Fos expression in dorsal horn nuclei of wild-type mice with intraplantar injection of SL-NH₂ (10 μg per paw) (WT-SL) (A) or LR-NH₂ (10 μg per paw) (WT-LR)(B), or TRPV1-deficient mice with intraplantar injection of SL-NH₂(10 μg per paw) (KO-SL) (C). D, Quantitative changes in Fos-LI in lamina I-II neurons of L4 and L5 dorsal horn after injection in WT-SL, W-LR, and KO-SL; n = 6 per group; *p < 0.05 versus WT-SL. Scale bar, 100 μm.