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. 2009 May 13;29(19):6217-28.
doi: 10.1523/JNEUROSCI.0893-09.2009.

TRPC1 and TRPC6 channels cooperate with TRPV4 to mediate mechanical hyperalgesia and nociceptor sensitization

Nicole Alessandri-Haber  1 , Olayinka A Dina, Xiaoje Chen, Jon D Levine
Affiliations

TRPC1 and TRPC6 channels cooperate with TRPV4 to mediate mechanical hyperalgesia and nociceptor sensitization

Nicole Alessandri-Haber et al. J Neurosci. .

Abstract

The transient receptor potential vanilloid 4 (TRPV4) contributes to mechanical hyperalgesia of diverse etiologies, presumably as part of a mechanoreceptor signaling complex (Alessandri-Haber et al., 2008). To investigate the hypothesis that a functional interaction between TRPV4 and stretch-activated ion channels (SACs) is involved in this mechanical transduction mechanism, we used a selective SACs inhibitor, GsMTx-4. Intradermal injection of GsMTx-4 in the rat hindpaw reversed the mechanical hyperalgesia induced by intradermal injection of inflammatory mediators. In vivo single fiber recordings showed that GsMTx-4 reversed inflammatory mediator-induced decrease in mechanical threshold in half of sensitized C-fibers. Furthermore, GsMTx-4 reduced hyperalgesia to both mechanical and hypotonic stimuli in different models of inflammatory and neuropathic pain, although it had no effect on baseline mechanical nociceptive thresholds. TRPC1 and TRPC6, two GsMTx-4-sensitive SACs, are expressed in dorsal root ganglion (DRG) neurons. Single-cell reverse transcription-PCR showed that messenger RNAs for TRPV4, TRPC1, and TRPC6 are frequently coexpressed in DRG neurons. Spinal intrathecal administration of oligodeoxynucleotides antisense to TRPC1 and TRPC6, like that to TRPV4, reversed the hyperalgesia to mechanical and hypotonic stimuli induced by inflammatory mediators without affecting baseline mechanical nociceptive threshold. However, antisense to TRPC6, but not to TRPC1, reversed the mechanical hyperalgesia induced by a thermal injury or the TRPV4-selective agonist 4alpha-PDD (4 alpha-phorbol 12,13-didecanoate). We conclude that TRPC1 and TRPC6 channels cooperate with TRPV4 channels to mediate mechanical hyperalgesia and primary afferent nociceptor sensitization, although they may have distinctive roles.

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Figures

Figure 1.
Figure 1.
GsMTx-4 inhibits mechanical hyperalgesia without affecting baseline mechanical nociceptive threshold. A, Evaluation of nociceptive mechanical threshold in the absence (black bars) or presence (white bars) of different doses of GsMTx-4. Up to a dose of 1000 ng, GsMTx-4 did not affect baseline nociceptive mechanical threshold. B, Intradermal injection of a soup of inflammatory mediators (PGE2, 5-HT, histamine, substance P, and bradykinin, 100 ng each) in rat hindpaw induces a decrease in mechanical nociceptive threshold (black bar). Mechanical nociceptive threshold was evaluated 30 min after administration of different doses of GsMTx-4 (white bars; 10, 100, and 1000 ng). GsMTx-4 (1000 ng) reversed inflammatory soup-induced mechanical hyperalgesia (*p < 0.001, Tukey's post hoc multiple comparison test). BL, Baseline.
Figure 2.
Figure 2.
GsMTx-4 reverses mechanical hyperalgesia associated with inflammation. A, Intradermal injection of the simplified inflammatory soup (PGE2 and 5-HT, 100 ng each) induces a decrease in mechanical threshold that was reversed by intradermal injection of GsMTx-4 (1000 ng) at the same site (116 ± 2 g, n = 12 for baseline; 72 ± 1 g, n = 12 after PGE2 and 5-HT; and 116 ± 2 g, n = 6 after GsMTx-4; *p < 0.001, Tukey's post hoc multiple comparison test). B, Intradermal injection of carrageenan (1%, 5 μl) in rat hindpaw induces a significant decrease in mechanical nociceptive threshold that was reversed by intradermal injection of GsMTx-4 (1000 ng) at the same site (118 ± 1 g for baseline, 75 ± 2 g after carrageenan, and 113 ± 3 g after GsMTx-4; n = 8; *p < 0.001, Tukey's post hoc multiple comparison test). The effect of GsMTx-4 is reversible; 24 h after its administration, the decrease in mechanical threshold was not significantly different from pre-GsMTx-4 baseline (p > 0.05, Tukey's post hoc multiple comparison test).
Figure 3.
Figure 3.
GsMTx-4 reverses the decrease in mechanical threshold induced by inflammatory soup in half of sensitized C-fibers. A, Intradermal injection of GsMTx-4 (1000 ng, 2.5 μl) adjacent to mechanical receptive fields did not induce significant activity in C-fibers. Inset, Mechanical thresholds of C-fibers were not significantly different from baseline after intradermal injection of GsMTx-4 (n = 13). B, Response of individual C-fibers to sustained (60 s) threshold mechanical stimulation before (●) and after (▴) intradermal injection of PGE2 and 5-HT and after a subsequent intradermal injection of GsMTx-4 (■). n is the number of fibers recorded. Injection of simplified inflammatory soup (PGE2 and 5-HT, 100 ng each) into the mechanical receptive field of C-fibers reduced the mechanical threshold in 15 of 26 C-fibers. A subsequent injection of GsMTx-4 (1000 ng, 2.5 μl) into the mechanical receptive field of a C-fiber reversed the decrease in mechanical thresholds induced by simplified soup in 50% of the sensitized C-fibers (8 of 15). C, There is no significant difference in the conduction velocity between C-fibers that were soup sensitive (n = 15) vs soup insensitive (n = 11) or between C-fibers that were GsMTx-4 sensitive or GsMTx-4 insensitive (p > 0.05, Tukey's post hoc multiple comparison test). D, Response of sensitized C-fibers to suprathreshold (10 g) mechanical stimulation before (●) and after (▴) intradermal injection of PGE2 and 5-HT and after a subsequent intradermal injection of GsMTx-4 (■). There was no significant difference between C-fibers that were GsMTx-4-sensitive or GsMTx-4-insensitive.
Figure 4.
Figure 4.
GsMTx-4 reverses paclitaxel chemotherapy-induced mechanical hyperalgesia. Rats treated with paclitaxel (daily for 10 d) develop mechanical hyperalgesia (118 ± 2 g, n = 12 before and 74 ± 3 g, n = 6 after paclitaxel; p < 0.001, Tukey's post hoc multiple comparison test). Intradermal injection of GsMTx-4 in the hindpaw 30 min before mechanical nociceptive threshold testing reversed the mechanical hyperalgesia (74 ± 3 g before GsMTx-4 and 116 ± 2 g after; n = 6; p < 0.001, Tukey's post hoc multiple comparison test). Twenty-four hours after the administration of GsMTx-4, mechanical nociceptive thresholds were not significantly different from pre-GsMTx-4 paw-withdrawal thresholds (n = 6 for each group; p > 0.05, Tukey's post hoc multiple comparison test).
Figure 5.
Figure 5.
GsMTx-4 also markedly reduces nociceptive flinching in response to hypotonic stimulation. A, Rats treated with PGE2 and 5-HT, carrageenan, or paclitaxel have, respectively, a 4-, 3.6-, and 3.2-fold increase in the number of nociceptive flinches in response to an intradermal injection of hypotonic solution (Hypo) (10 μl of deionized water, 17 mOsm) compared with control rats. GsMTx-4 markedly reduced the number of flinches in PGE2 and 5-HT-treated (16 ± 2, n = 12 before and 8 ± 1, n = 6 after GsMTx-4), carrageenan-treated (17 ± 1, n = 12 before and 6 ± 1, n = 8 after GsMTx-4), and paclitaxel-treated (13 ± 2, n = 10 before and 7 ± 1, n = 6 after GsMTx-4) rats. All asterisks, p < 0.05, Tukey's post hoc multiple comparison test. Twenty-four hours after the administration of GsMTx-4, its effect was no longer present, and there was no significant difference in the number of flinches between the different groups of rats (p > 0.05, Tukey's multiple comparison test). B, Small-diameter (≤25 μm) DRG neurons from TRPV4+/+ and TRPV4−/− mice were first challenged with a 30% hypotonic solution containing PGE2 and 5-HT (10 μm each) for 3 min and then challenged with a 30% hypotonic solution containing PGE2, 5-HT, and GsMTx-4 (500 nm) for 3 min. The increase in [Ca2+]i induced by the hypotonic solution containing PGE2 and 5-HT is reduced in the presence of GsMTx-4 in TRPV4+/+ mice (2.4 ± 0.2 μm before and 1.7 ± 0.2 μm after GsMTx-4; n = 24; p = 0.007, paired Student's t test) as well as in TRPV4−/− mice (1.7 ± 0.1 μm before and 1.4 ± 0.1 μm after GsMTx-4; n = 14; p = 0.03, paired Student's t test). C, Dissociated lumbar DRG neurons were cultured for 2 d. Cells were scraped in homogenization buffer, and subcellular fractionation was performed. The membrane fraction was separated on an electrophoresis gel and transferred to a polyvinylidene difluoride membrane. The membrane was then probed in parallel with anti-TRPV4 (1:500), anti-TRPC1 (1:500), and anti-TRPC6 (1:500) antibodies. The expected bands at 120 kDa for TRPC1, 110 kDa for TRPC6, and the doublet bands at 98 and 107 kDa for TRPV4 were detected. D, The cytoplasm of 47 DRG neurons was harvested after 2 d in culture, and multiplex single-cell RT-PCR was performed. Neurons expressed from none to all three of the mRNAs of interest; here we show the example of neurons expressing mRNA for TRPC1 (168 bp) or TRPC1 and TRPC6 (228 bp) or TRPC1, TRPC6, and TRPV4 (221 bp).
Figure 6.
Figure 6.
Hyperalgesia to mechanical and hypotonic stimuli induced by PGE2 and 5-HT is TRPC1 and TRPC6 dependent. A, Treatment with TRPC1 or TRPC6 antisense (AS) ODN for 3 d did not affect baseline nociceptive mechanical threshold in control rats (n = 24 for baseline and n = 6 for antisense and mismatch-treated rats; p > 0.05, Tukey's post hoc multiple comparison test). In contrast, the mechanical hyperalgesia induced by PGE2 and 5-HT was reversed by treatment with TRPC1 or TRPC6 antisense compared with mismatch-treated (MM) rats (n = 18 for baseline and n = 6 for antisense and mismatch-treated rats; *p < 0.001, Tukey's post hoc multiple comparison test). The effect of the antisense was reversible; 4 d after the last ODN injection, the mechanical hyperalgesia induced by PGE2 and 5-HT was not significantly different from pre-ODN baseline (p > 0.05, Tukey's multiple-comparison test). B, Treatment with TRPC1 or TRPC6 antisense also markedly reduced the number of flinches in response to intradermal injection of hypotonic solution (Hypo) in the presence of PGE2 and 5-HT compared with mismatch-treated rats (n = 6 for hypotonic solution in presence of PGE2 plus 5-HT and n = 6 for each ODN group; *p < 0.001, Tukey's post hoc multiple comparison test). C, Paw-withdrawal thresholds were markedly decreased 24 h after thermal injury (118 ± 1 g before vs 74 ± 1 g after thermal injury; n = 36; p < 0.0001, paired Student's t test). Treatment with TRPV4 or TRPC6 antisense for 3 d markedly reversed the mechanical hyperalgesia compared with mismatch ODN-treated rats (n = 6 for all groups). Again, the effect of the antisense treatment was reversible; there was no significant difference in the mechanical thresholds between baseline and ODN-treated groups 4 d after the last ODN injection (n = 36 for baseline and n = 6 for each ODN group; all p > 0.05, Tukey's post hoc multiple comparison test). In contrast, TRPC1 antisense did not affect the mechanical hyperalgesia induced by thermal injury. D, There was a 37 ± 4 and 32 ± 7% decrease in TRPC1 and TRPC6 protein expression level, respectively (n = 3 for all groups; p < 0.05, unpaired Student's t test) in DRG of rats treated with antisense (AS) ODN compared with mismatch ODN-treated rats (MM). The amount of protein loaded in each lane was normalized by probing the membrane with an anti-GAPDH antibody.
Figure 7.
Figure 7.
Antisense oligodeoxynucleotides to TRPC6 reverses 4α-PDD-induced hyperalgesia to mechanical and hypotonic stimuli. A, Intradermal injection of 4 α-PDD (1 μg/2.5 μl) in rat hindpaw 30 min before measurement of nociceptive thresholds induced a significant decrease in mechanical thresholds (120 ± 1 g, n = 8 for baseline vs 76 ± 1 g,; n = 8 after 4α-PDD; *p < 0.0001, paired Student's t test). In contrast, intradermal injection of the vehicle, 10% DMSO, had no significant effect on mechanical thresholds (n = 4 before and after 10% DMSO; p > 0.05, paired Student's t test). BL, Baseline. B, Treatment with TRPC6 antisense (AS) ODN for 3 d reversed the mechanical hyperalgesia induced by 4α-PDD compared with mismatch-treated (MM) rats (n = 8 for baseline and n = 6 for each ODN group; *p < 0.001, Tukey's post hoc multiple comparison test). The effect of the antisense was reversible; 4 d after the last ODN injection, the mechanical thresholds was similar in baseline and ODN-treated rats (p > 0.05, Tukey's multiple comparison test). In contrast, treatment with TRPC1 antisense ODN does not affect the decrease in mechanical threshold induced by 4α-PDD (p > 0.05, Tukey's post hoc multiple comparison test). C, Intradermal injection of 4α-PDD in rat hindpaw induces a significant number of flinches (15 ± 1, n = 6). Moreover, rats pretreated with 4α-PDD have a sixfold increase in the number of nociceptive flinches in response to an injection of hypotonic solution (4 ± 1, n = 12 for hypotonic in the absence and 24 ± 3, n = 10 in the presence of 4α-PDD; p < 0.0001, unpaired Student's t test). Treatment with TRPC6 antisense ODN markedly reduced the number of flinches induced by intradermal injection of hypotonic solution in the presence of 4α-PDD compared with mismatch-treated rats (24 ± 3, n = 10 for baseline 4α-PDD plus hypotonic solution; 8 ± 1, n = 6 for antisense-treated rats; and 20 ± 1, n = 6 for mismatch-treated rats; *p < 0.001, Tukey's post hoc multiple comparison test). Again the effect of antisense was reversible; 4 d after the last ODN injection, the number of flinches was not significantly different between TRPC6 antisense- and mismatch-treated rats. In contrast, antisense ODN to TRPC1 did not affect the number of flinches (n = 10 for baseline 4α-PDD plus hypotonic solution and n = 6 for ODN groups; p > 0.05, Tukey's post hoc multiple comparison test).
Figure 8.
Figure 8.
Inflammatory mediators engage TRPV4, TRPC6, and TRPC1 channels in mechanical transduction. Sensing of osmotic or mechanical stimuli by the phospholipid bilayer leads to the activation of membrane-bound G-proteins and PLC and/or activation of phospholipase A2 (PLA2), which generates 5′,6′-epoxyeicosatrienoic acid (5′,6′-EET) via the metabolism of arachidonic acid (AA), which in turn acts as a TRPV4 agonist. Integrins also detect osmotic and mechanical stimuli and activate Src protein tyrosine kinase family (Src PTK), which can directly activate cAMP/protein kinase A (PKA), PLC/protein kinase C (PKC) phosphorylation pathways. Inflammatory mediators bind to their respective GPCRs, which also activate PLC and cAMP/PKA phosphorylation pathways. Activation of PLC generates IP3, which acts on IP3Rs to induce calcium release. IP3Rs can directly interact with TRPV4 and TRPC1 to activate/sensitize these channels. Activation of the different phosphorylation pathways will sensitize or desensitize TRPV4, TRPC6, and TRPC1. Putative heteromeric channels (?) that would further increase the complexity cannot be ruled out. Light gray lines represent processes engaging TRPV4, dark gray lines represent processes engaging TRPC1, and gray lines represent processes engaging TRPC6. AC, Adenylate cyclase; DAG, diacylglycerol; G, G protein; PIP2, phosphatidylinositol-4, 5-biphosphate.
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