Transient receptor potential vanilloid 4 is essential in chemotherapy-induced neuropathic pain in the rat

Abstract

The development of treatments for neuropathic pain has been hindered by our limited understanding of the basic mechanisms underlying abnormalities in nociceptor hyperexcitability. We recently showed that the polymodal receptor transient receptor potential vanilloid 4 (TRPV4), a member of the transient receptor potential (TRP) family of ion channels, may play a role in inflammatory pain (Alessandri-Haber et al., 2003). The present study tested whether TRVP4 also contributes to neuropathic pain, using a rat model of Taxol-induced painful peripheral neuropathy. Taxol is the most widely used drug for the treatment of a variety of tumor types, but the dose of Taxol that can be tolerated is limited by the development of a small-fiber painful peripheral neuropathy. We found that Taxol treatment enhanced the nociceptive behavioral responses to both mechanical and hypotonic stimulation of the hind paw. Spinal administration of antisense oligodeoxynucleotides to TRPV4, which reduced the expression of TRPV4 in sensory nerve, abolished Taxol-induced mechanical hyperalgesia and attenuated hypotonic hyperalgesia by 42%. The enhancement of osmotic nociception involves sensitization of osmotransduction in primary afferents because osmotransduction was enhanced in cultured sensory neurons isolated from Taxol-treated rats. Taxol-induced TRPV4-mediated hyperalgesia and the enhanced osmotransduction in cultured nociceptors were dependent on integrin/Src tyrosine kinase signaling. These results suggest that TRPV4 plays a crucial role in a painful peripheral neuropathy, making it a very promising target for the development of a novel class of analgesics.

Figure 1.

Taxol-induced mechanical hyperalgesia and hypotonicity-induced nociception are mediated by TRPV4. A, Taxol-induced mechanical hyperalgesia was abolished by TRPV4 antisense ODN treatment, whereas in mismatch-treated rats the mechanical hyperalgesia was not statistically different from the baseline (mean ± SEM; –0.8 ± 1.3gm, n = 8 for antisense-treated vs 33.5 ± 1.4 gm, n = 8 for mismatch-treated rats; p < 0.05, unpaired Student's t test). At 4 d after the last antisense ODN treatment the Taxol-induced mechanical hyperalgesia was not statistically different from the pre-ODN baseline (p > 0.05, Tukey's multiple comparison test; n = 8). B, Injection of 10 μl of hypotonic solution induced a significant number of flinches in Taxol-treated rats as compared with isotonic solution (11.6 ± 1.1, n = 16 for hypotonic vs 0.4 ± 0.2, n = 8 for isotonic solution; p < 0.05, unpaired Student's t test). The hypotonicity-induced number of flinches was significantly higher in Taxol-treated rats versus control rats (11.6 ± 1.1, n = 16 for Taxol-treated vs 3.2 ± 0.3, n = 18 for control rats; p < 0.05, unpaired Student's t test). C, Effect of TRPV4 antisense on the number of flinches induced by the injection of 10 μl of hypotonic solution in the hind paw of Taxol-treated rats. Spinal intrathecal administration of TRPV4 antisense decreased the number of hypotonicity-induced flinches by 42% as compared with mismatch-treated rats (6 ± 1, n = 6 for the antisense-treated vs 10.5 ± 1.1, n = 8 for the mismatch-treated rats; p = 0.01, unpaired Student's t test). At 4 d after the last ODN treatment there were no significant differences in the number of hypotonicity-induced flinches between the two ODN groups (mean ± SEM; 8.5 ± 1.2, n = 6 for the antisense-treated vs 9.1 ± 1.3, n = 8 for the mismatch-treated rats; p > 0.05, unpaired Student's t test). D, A doublet band (98 and 107 kDa) was detected by Western blot in the saphenous nerve of Taxol-treated rats. There was a 55% ± 17 decrease in the level of TRPV4 protein in saphenous nerve from antisense-treated versus mismatch-treated rats (p < 0.05, unpaired Student's t test; n = 5 for antisense and n = 6 for mismatch). The amount of protein in both lanes was confirmed to be comparable (16.8 μg/lane) by reprobing the membrane with an α-tubulin antibody.

Figure 2.

The enhanced osmotransduction in cultured nociceptors of Taxol-treated rats does not depend on an increase in the expression level of TRPV4 protein. A, Example of the hypotonicity-induced increase in fluorescence ratio in a neuron challenged with a 30% hypotonic solution (212 mOsm). The excitability of the neuron was confirmed by a short exposure to KCl (20 mm), and the neuron was identified as a putative nociceptor when capsaicin (1 μm) induced a significant increase in the fluorescence ratio. B, The percentage of nociceptors responding to a 30% hypotonic challenge with an increase in the fluorescence ratio in Taxol-treated rats is significantly higher than that in controls (p < 0.025, χ² test with Yates correction). C, There was no significant difference in the level of TRPV4 protein in saphenous nerve from control and Taxol-treated rats (p > 0.05, unpaired Student's t test; n = 7 for control and n = 12 for Taxol-treated rats). The amount of protein in both lanes was confirmed to be comparable by reprobing the membrane with an α-tubulin antibody.

Figure 3.

TRPV4-mediated enhanced nociception is integrin-dependent. A, Effect of integrin antagonist peptide GRGDTP or its inactive analog GRADSP (1 μg/2.5 μl) on mechanical hyperalgesia in Taxol-treated rats. Injection of GRGDTP 30 min before the evaluation of mechanical nociceptive threshold reversed Taxol-induced mechanical hyperalgesia (mean ± SEM; 0.45 ± 2.1 gm, n = 6 after vs 35.6 ± 1.7 gm, n = 12 before GRGDTP; *p < 0.05, unpaired Student's t test), whereas GRADSP had no significant effect (mean ± SEM; 34 ± 2.1 gm, n = 6 after vs 35.6 ± 1.7 gm, n = 12 before GRADSP; p > 0.05, unpaired Student's t test). At 4 d after treatment with either GRGDTP or GRADSP peptide the mechanical threshold was not significantly different from the baseline (p > 0.05, Tukey's multiple comparison test). B, Effect of GRGDTP and its inactive analog GRADSP (1 μg/2.5 μl) on the number of hypotonicity-induced flinches in Taxol-treated rats. Injection of GRGDTP peptide 45 min before the hypotonic stimulation inhibited by 50% the number of hypotonicity-induced flinches (mean ± SEM; 5.7 ± 0.9, n = 6 after vs 11.6 ± 1.6, n = 12 before GRGDTP; *p < 0.05, unpaired Student's t test), whereas GRADSP had no significant effect (mean ± SEM; 12.6 ± 1.6, n = 5 after vs 11.6 ± 1.6, n = 12 before GRADSP; p > 0.05, unpaired Student's t test). At 4 d after the peptide treatment there were no significant differences between the baseline number of flinches induced by hypotonicity and the number of flinches in GRGDTP- and GRADSP-treated rats (p > 0.05, Tukey's multiple comparison test). C, Mean of normalized fluorescence ratio ± SEM when nociceptors are challenged with hypotonic solution in the absence or presence of GRGDTP or GRADSP peptide (10 μm). Presence of GRGDTP during the hypotonic stimulus inhibited the hypotonicity-induced increase in fluorescence ratio by 88% (normalized fluorescence ratio was 1.19 ± 0.05 without vs 1.05 ± 0.02 in presence of GRGDTP; n = 8; *p < 0.05, paired Student's t test), whereas GRADSP had no significant effect (1.08 ± 0.01 before vs 1.11 ± 0.02 after GRADSP; n = 9; p > 0.05, paired Student's t test). Data are reported as the fluorescence ratio amplitude of the effect of hypotonic in the absence or presence of RGD peptide normalized to the fluorescence ratio obtained in isotonic solution in the absence or presence of RGD peptide. D, Mean of normalized fluorescence ratio ± SEM for nociceptors stimulated by different extracellular solutions containing DIDS (100 μm). Nociceptors from control rats were challenged first with the direct TRPV4 activator 4 α-PDD, after full recovery of [Ca²⁺]_i. Nociceptors were challenged with 30% hypotonic solution and perfused with isotonic solution until [Ca²⁺]_i recovery. Finally, nociceptors were perfused for 20 min with an isotonic solution containing GRGDTP (50 μm) and challenged with a 30% hypotonic solution containing GRGDTP for 3 min. Only nociceptors responding to both 30% hypotonic and 4 α-PDD stimuli were included in the graph (n = 10). The mean fluorescence ratio during 4α-PDD challenge was not significantly different from the one during hypotonic (1.15 ± 0.03 during 4 α-PDD vs 1.14 ± 0.03 during hypotonic stimulation; p > 0.05, paired Student's t test; n = 10). The presence of GRGDTP during the hypotonic stimulation significantly inhibited the hypotonicity-induced increase in fluorescence ratio (1.14 ± 0.03 during hypotonic alone vs 1.05 ± 0.03 during hypotonic in the presence of GRGDTP; *p < 0.05, paired Student's t test; n = 10). Inset, TRPV4 mRNA (428 bp) was detectable by single-cell RT-PCR in the responsive nociceptors.

Figure 4.

TRPV4-mediated enhanced nociception is Src tyrosine kinase-dependent. A, Effect of tyrosine kinase inhibitors on mechanical hyperalgesia in Taxol-treated rats. Injection of PP₁ (Src family kinase-specific inhibitor; 1 μg/2.5 μl) and genistein (general tyrosine kinase inhibitor; 1 μg/2.5 μl) 30 min before the evaluation of mechanical nociceptive threshold reversed Taxol-induced mechanical hyperalgesia (mean ± SEM; 1.75 ± 2.3, n = 6 for PP₁-treated rats; –1.61 ± 2.4, n = 6 for the genistein-treated rats; 36.5 ± 0.9, n = 46 for the baseline). Piceatannol (specific inhibitor of Syk, a related cytoplasmic tyrosine kinase; 1 μg/2.5 μl) did not have any significant effect (mean ± SEM; 38.4 ± 1.6, n = 6 for piceatannol-treated rats vs 36.5 ± 0.9, n = 46 for the baseline; p > 0.05, unpaired Student's t test). At 24 hr after inhibitor injection there were no significant differences among the three groups of rats (mean ± SEM; 11.2 ± 1.8, n = 6 after recovery from PP₁, 12.8 ± 1.4, n = 6 after piceatannol, and 10.3±2.1 after recovery from genistein treatment; p > 0.05, Tukey's multiple comparison test). B, Effect of tyrosine kinase inhibitors on the number of hypotonicity-induced flinches. Injection of PP₁ or genistein in Taxol-treated rat hind paw 45 min before hypotonic stimulation inhibited the number of flinches by 44% (mean ± SEM; 6.5 ± 0.8, n = 6 for PP₁-treated vs 5.3 ± 1.1, n = 6 for the genistein-treated rats), whereas piceatannol did not significantly affect the number of flinches (mean ± SEM; 10.7 ± 1.8, n = 6 for piceatannol-treated vs 11.6 ± 1.6, n = 18 for the baseline number of flinches; p > 0.05, unpaired Student's t test). At 24 hr after the injection of inhibitors there were no significant differences in the number of flinches among the groups of rats (p > 0.05, Tukey's multiple comparison test). C, Mean of normalized fluorescence ratio for nociceptors, in vitro, first challenged with a 30% hypotonic solution and then challenged with a 30% hypotonic solution containing either PP₁ or piceatannol (10 μm). PP₁ prevented the hypotonicity-induced increase in fluorescence ratio (1.06 ± 0.01, n = 5 before vs 0.99 ± 0.01, n = 5 after PP₁; *p < 0.05, paired Student's t test), whereas the addition of piceatannol did not have any effect (1.06 ± 0.01, n = 8 before vs 1.11 ± 0.02, n = 8 after piceatannol; p > 0.05, paired Student's t test).