A transient receptor potential vanilloid 4-dependent mechanism of hyperalgesia is engaged by concerted action of inflammatory mediators

Abstract

The transient receptor potential vanilloid 4 (TRPV4) is a primary afferent transducer that plays a crucial role in neuropathic hyperalgesia for osmotic and mechanical stimuli, as well as in inflammatory mediator-induced hyperalgesia for osmotic stimuli. In view of the clinical importance of mechanical hyperalgesia in inflammatory states, the present study investigated the role of TRPV4 in mechanical hyperalgesia induced by inflammatory mediators and the second-messenger pathways involved. Intradermal injection of either the inflammogen carrageenan or a soup of inflammatory mediators enhanced the nocifensive paw-withdrawal reflex elicited by hypotonic or mechanical stimuli in rat. Spinal administration of TRPV4 antisense oligodeoxynucleotide blocked the enhancement without altering baseline nociceptive threshold. Similarly, in TRPV4(-/-) knock-out mice, inflammatory soup failed to induce any significant mechanical or osmotic hyperalgesia. In vitro investigation showed that inflammatory mediators engage the TRPV4-mediated mechanism of sensitization by direct action on dissociated primary afferent neurons. Additional behavioral observations suggested that multiple mediators are necessary to achieve sufficient activation of the cAMP pathway to engage the TRPV4-dependent mechanism of hyperalgesia. In addition, direct activation of protein kinase A or protein kinase C epsilon, two pathways that mediate inflammation-induced mechanical hyperalgesia, also induced hyperalgesia for both hypotonic and mechanical stimuli that was decreased by TRPV4 antisense and absent in TRPV4(-/-) mice. We conclude that TRPV4 plays a crucial role in the mechanical hyperalgesia that is generated by the concerted action of inflammatory mediators present in inflamed tissues.

Figure 1.

TRPV4 mediates inflammatory hyperalgesia. A, Inflammatory mediators enhance nocifensive flinching induced by the injection of hypotonic solution. Intradermal injection of a hypotonic solution in rat hindpaw induced nocifensive flinches. Injection of carrageenan (Carr) or inflammatory soup (Soup) before the hypotonic solution induced a 4.6- and a 3.6-fold increase, respectively, in the number of flinches. Treatment with TRPV4 antisense ODN reduced the number of hypotonicity-induced flinches in the presence of carrageenan by 72% (5 ± 1, n = 6 for antisense-treated vs 18 ± 2, n = 6 for mismatch-treated rats; *p < 0.05, unpaired Student's t test) and the ones in the presence of the inflammatory soup by 43% (9 ± 2, n = 6 for antisense-treated vs 16 ± 2, n = 6 for mismatch-treated rats; *p < 0.02, unpaired Student's t test). B, Intradermal injection of carrageenan or inflammatory soup induced a decrease in mechanical nociceptive thresholds in the rat. Spinal intrathecal injection of TRPV4 antisense ODN prevented the mechanical hyperalgesia induced by carrageenan (3.5 ± 3%, n = 6 for antisense-treated vs 26 ± 0.8%, n = 6 for mismatch-treated rats; *p < 0.001, unpaired Student's t test) or by inflammatory soup (−1 ± 4%, n = 6 for antisense-treated vs 30 ± 2%, n = 6 for mismatch-treated rats; *p < 0.0001, unpaired Student's t test). Inset, Treatment with TRPV4 antisense had no effect on mechanical threshold in naive rats (n = 58 for mechanical threshold baseline and n = 20 for each ODN-treated rats; p > 0.05, ANOVA), whereas it prevented the decrease in threshold caused by inflammatory soup or carrageenan.

Figure 2.

Inflammatory soup does not induce mechanical hyperalgesia in TRPV4^−/− mice. A, The baseline withdrawal response frequency to von Frey hair stimulation was not significantly different between TRPV4^+/+ and TRPV4^−/− mice (n = 10 for TRPV4^+/+ and n = 12 for TRPV4^−/− mice; *p > 0.05, unpaired Student's t test). However, in the presence of inflammatory soup, the withdrawal response frequency was increased by 2.8-fold in TRPV4^+/+ mice (30 ± 5% before and 84 ± 3% after inflammatory soup; n = 10; p < 0.0001, paired Student's t test), whereas the response remained unchanged in TRPV4^−/− mice (n = 12; p > 0.05, paired Student's t test). B, Small-diameter DRG neurons from TRPV4^+/+ and TRPV4^−/− mice were first challenged with a 30% hypotonic solution for 3 min and then challenged with a 30% hypotonic solution containing the inflammatory soup (bradykinin, histamine, serotonin, substance P, and PGE₂, 10 μm each). C, In TRPV4^+/+ mice, the increase in [Ca²⁺]_i induced by a 30% hypotonic challenge was significantly enhanced in the presence of inflammatory soup (1.7 ± 0.1 μm before and 2.3 ± 0.2 μm after the soup; n = 23; p < 0.05, paired Student's t test). In contrast, in TRPV4^−/− mice, the response of DRG neurons to hypotonicity was unaffected (n = 21; p > 0.05, paired Student's t test). Iso, Isotonic solution; caps, capsaicin.

Figure 3.

TRPV4 participation in mechanical hyperalgesia requires a threshold level of cAMP reached only by combined mediators. A, Injection of 1 μg of PGE₂ in rat hindpaw induced a slightly greater mechanical hyperalgesia compared with the injection of 100 ng of PGE₂ (mean ± SEM; 32 ± 0.7%, n = 48 for 100 ng of PGE₂ vs 37 ± 1%, n = 12 for 1 μg of PGE₂; p < 0.001, unpaired Student's t test). However, treatment with antisense ODN for TRPV4 did not reduce the100 ng nor the 1 μg PGE₂-induced mechanical hyperalgesia (n = 26 for antisense- and mismatch-treated for 100 ng of PGE₂ and n = 6 for 1 μg PGE₂ antisense- and mismatch-treated rats; p > 0.05, unpaired Student's t test). Injection of 100 ng or 1 μg of 5-HT in rat hindpaw induced a similar level of mechanical hyperalgesia (n = 6; p > 0.05, unpaired Student's t test). Again, treatment with antisense ODN for TRPV4 did not reduce the 5-HT-induced mechanical hyperalgesia (n = 6 for each ODN group; p > 0.05, unpaired Student's t test). In contrast, treatment with antisense ODN for TRPV4 prevented the decrease in paw-withdrawal threshold induced by the coinjection of PGE₂ and 5-HT (100 ng each; 0.6 ± 1.5%, n = 8 for antisense-treated and 33 ± 1%, n = 8 for mismatch-treated rats; *p < 0.05, unpaired Student's t test). B, Intraplantar injection of PGE₂ or 5-HT (100 ng and 1 μg each) similarly enhanced the withdrawal response frequency to mechanical stimuli in both TRPV4^+/+ and TRPV4^−/− mice. In TRPV4^+/+ mice, the coinjection of PGE₂ and 5-HT (100 ng each) induced a 3.9-fold increase in the withdrawal frequency response (23 ± 3%, n = 18 before and 90 ± 4%, n = 6 after coinjection of PGE₂ and 5-HT). In contrast, in TRPV4^−/− mice, the withdrawal response frequency after the coinjection of PGE₂ and 5-HT was not different from baseline (n = 6 after and n = 23 before treatment; *p > 0.05, unpaired Student's t test).

Figure 4.

PGE₂-induced hyperalgesia switches to a TRPV4-dependent mechanism when a sufficient level of cAMP is reached. A, Rats were treated with either TRPV4 antisense or mismatch ODN for 3 d, and mechanical nociceptive thresholds were evaluated before and 15 min after injection of increasing doses of 8-Br-cAMP (0.1, 0.3, 1, 3, 10, 100, 300, and 1000 ng). The mechanical hyperalgesia induced by 10–1000 ng of 8-Br-cAMP was reduced in TRPV4 antisense-treated compared with mismatch-treated rats. B, Intradermal injection of 1 μg of PGE₂ induced a significant decrease in mechanical threshold. The injection of 1 ng of 8-Br-cAMP had no effect on mechanical nociceptive threshold and injection of 1 ng of 8-Br-cAMP 30 min before the injection of 1 μg of PGE₂ did not enhance PGE₂-induced mechanical hyperalgesia (37 ± 1%, n = 12 for PGE₂ alone and 34 ± 2%, n = 12 for 8-Br-cAMP plus PGE₂; p > 0.05, unpaired Student's t test). However, in the presence of 8-Br-cAMP, treatment with TRPV4 antisense ODN prevented the PGE₂-induced mechanical hyperalgesia (3 ± 2%, n = 12 for antisense-treated vs 36 ± 2%, n = 8 for mismatch-treated rats; *p < 0.0001, unpaired Student's t test). C, Intraplantar injection of 1 ng of 8-Br-cAMP did not affect the withdrawal response frequency in TRPV4^+/+ and TRPV4^−/− mice and injection of 100 ng of PGE₂ induced a threefold increase in the withdrawal response frequency in both TRPV4 genotypes. In contrast, after pretreatment with 1 ng of 8-Br-cAMP, the mechanical hyperalgesia induced by 100 ng of PGE₂ was absent in TRPV4^−/− mice (n = 12). Furthermore, in the presence of the inhibitor of activation by cAMP of cAMP-dependent protein kinase I and II, Rp-cAMPS, coinjection of PGE₂ and 5-HT (100 ng each) failed to increase the withdrawal response frequency in TRPV4^+/+ mice (27 ± 6%, n = 12 for PGE₂ plus 5-HT plus Rp-cAMPS compared with 70 ± 4%, n = 6 for PGE₂ plus 5-HT; *p < 0.05, unpaired Student's t test).

Figure 5.

TRPV4 participates in cAMP/PKA-mediated hyperalgesia. A, 8-Br-cAMP and PKACS induced, respectively, a threefold and a fourfold increase in the number of flinches induced by hypotonicity in rat (12 ± 1, n = 14 after 8-Br-cAMP, 15 ± 1, n = 10 after PKACS vs 3.9 ± 0.3, n = 24 for hypotonicity alone). Treatment with TRPV4 antisense ODN reduced the number of flinches induced by hypotonicity in the presence of 8-Br-cAMP or PKACS, respectively, by 41% (7.2 ± 0.8, n = 8 for antisense-treated vs 12.2 ± 0.9, n = 8 for mismatch-treated rats; *p < 0.01, unpaired Student's t test) and 48% (8 ± 1, n = 8 for antisense-treated vs 16 ± 2, n = 7 for mismatch-treated rats; *p = 0.0001, unpaired Student's t test). B, Intraplantar injection of PKACS induced a 2.5-fold increase in the withdrawal response frequency in TRPV4^+/+ mice (25 ± 6% before and 63 ± 54% in the presence of PKACS; n = 8; *p < 0.001, paired Student's t test). In contrast, PKACS did not affect the withdrawal response frequency in TRPV4^−/− mice (n = 18; p > 0.05, paired Student's t test). C, Injection of 8-Br-cAMP or PKACS in rat hindpaw induced a similar degree of mechanical hyperalgesia (35 ± 2%, n = 6 for PKACS vs 35 ± 1%, n = 17 for 8-Br-cAMP; *p > 0.05, unpaired Student's t test). Treatment with TRPV4 antisense ODN attenuated both the 8-Br-cAMP- (4.9 ± 2.6%, n = 8 for antisense-treated vs 35.7 ± 1.3%, n = 8 for mismatch-treated rats; *p < 0.0001, unpaired Student's t test) and the PKACS-induced mechanical hyperalgesia (−3.9 ± 3.8%, n = 8 for antisense-treated vs 32.7 ± 2.5%, n = 8 for mismatch-treated rats; *p < 0.0001, unpaired Student's t test). In contrast, mismatch treatment had no effect on the decrease in mechanical threshold caused by PKACS or 8-Br-cAMP. D, Mean ± SEM of [Ca²⁺]_i for DRG neurons first challenged with a 30% hypotonic solution and then challenged with a 30% hypotonic solution containing either Rp-cAMPS (30 μm) or H89 (10 μm). In TRPV4^+/+ mice, the hypotonicity-induced increase in [Ca²⁺]_i was significantly reduced in the presence of Rp-cAMPS or H89 (1.2 ± 0.1 μm, n = 4 for Hypo plus H89, 1.3 ± 0.08 μm, n = 8 for Hypo plus Rp-cAMPS vs 1.7 ± 0.1 μm, n = 12 for Hypo; *p < 0.05, Tukey's multiple comparison test). In contrast, these inhibitors did not significantly affect the response of DRG neurons to hypotonicity in TRPV4^−/− mice (p > 0.05, ANOVA and Tukey's multiple comparison test).

Figure 6.

TRPV4 participates in PKCϵ-mediated hyperalgesia. A, The number of flinches induced by hypotonic stimulation was 3.3-fold higher in the presence of the PKCϵ activator ψϵ-RACK (13 ± 2, n = 6 after ψϵ-RACK vs 3.9 ± 0.3, n = 24 before; p < 0.0001, unpaired Student's t test). Treatment with TRPV4 antisense reduced the number of flinches by 46% compared with mismatch-treated rats (5 ± 0.4, n = 6 for antisense-treated vs 11 ± 2, n = 6 for mismatch-treated rats; *p < 0.02, unpaired Student's t test). B, Mean ± SEM of [Ca²⁺]_i. DRG neurons from TRPV4^+/+ and TRPV4^−/− mice were first challenged with a 30% hypotonic solution and then challenged with a 30% hypotonic solution containing the PKCϵ translocation inhibitor peptide (2 μm). The hypotonicity-induced increase in [Ca²⁺]_i was significantly reduced in the presence of PKCϵ translocation inhibitor peptide in TRPV4^+/+ mice (1.3 ± 0.01 μm in the presence of PKCϵ inhibitor vs 1.7 ± 0.1 μm without; n = 13; *p < 0.001, paired Student's t test). In contrast, the inhibitor had no effect on the response of DRG neurons to hypotonicity in TRPV4^−/− mice (n = 12; p > 0.05, paired Student's t test). C, Treatment with TRPV4 antisense attenuated the decrease in paw-withdrawal threshold induced by the injection of ψϵ-RACK (1 μg, −0.03 ± 2.10%, n = 6 for antisense-treated vs 28.2 ± 1.7%, n = 6 for mismatch-treated rats; *p < 0.0001, unpaired Student's t test). D, Intraplantar injection of ψϵ-RACK increased the withdrawal response frequency by 2.6-fold in TRPV4^+/+ mice (32 ± 5% before and 83 ± 4% in the presence of ψϵ-RACK; n = 12; *p < 0.0001, unpaired Student's t test). In contrast, it did not affect the response in TRPV4^−/− mice (n = 14; p > 0.05, unpaired Student's t test).