Mechanosensitive TRPV4 Channel-Induced Extracellular ATP Accumulation at the Acupoint Mediates Acupuncture Analgesia of Ankle Arthritis in Rats

Abstract

(1) Background: Acupuncture (AP) is a safe and effective analgesic therapy. Understanding how fine needles trigger biological signals can help us optimize needling manipulation to improve its efficiency. Adenosine accumulation in treated acupoints is a vital related event. Here, we hypothesized that extracellular ATP (eATP) mobilization preceded adenosine accumulation, which involved local activation of mechanosensitive channels, especially TRPV4 protein. (2) Methods: AP was applied at the injured-side Zusanli acupoint (ST36) of acute ankle arthritis rats. Pain thresholds were assessed in injured-side hindpaws. eATP in microdialysate from the acupoints was determined by luminescence assay. (3) Results: AP analgesic effect was significantly suppressed by pre-injection of GdCl₃ or ruthenium red in ST36, the wide-spectrum inhibitors of mechanosensitive channels, or by HC067047, a specific antagonist of TRPV4 channels. Microdialysate determination revealed a needling-induced transient eATP accumulation that was significantly decreased by pre-injection of HC067047. Additionally, preventing eATP hydrolysis by pre-injection of ARL67156, a non-specific inhibitor of ecto-ATPases, led to the increase in eATP levels and the abolishment of AP analgesic effect. (4) Conclusions: These observations indicate that needling-induced transient accumulation of eATP, due to the activation of mechanosensitive TRPV4 channels and the activities of ecto-ATPases, is involved in the trigger mechanism of AP analgesia.

Keywords: TRPV4; acupuncture analgesia; extracellular ATP; mechano-sensitivity; nucleotidases.

Figure 1

Timeline of the behavioral measurements. Before the tests, rats were allowed to acclimate for 3 days. CFA was administrated on day 0. AP was performed two days later. Pain thresholds were determined just before CFA injection (baseline) and before (modeling) and after treatment. Beh. Test: Behavioral test. Acup: Acupuncture treatment.

Figure 2

Effects of AP treatment on CFA-induced pain hypersensitivity (n = 6–9, mean ± SE). (a,b) Changes in tactile allodynia (PWT) and thermal hyperalgesia (PWL) of injured-side hindpaws, respectively. ### p < 0.001 vs. control. *** p < 0.001 vs. model.

Figure 3

Involvement of MS channels at the acupoints in the AP analgesic mechanism (n = 4–9, mean ± SE). (a,b) Effects of pre-injection of GdCl₃ (1 mM, 20 μL) in ST36 on AP-relieved PWT and PWL, respectively. (c,d) Effects of pre-injection of RuR (0.5 mM, 20 μL) in ST36 on AP-relieved PWT and PWL, respectively. Data illustrate the final behavioral tests. Numbers above each column denote sample size (n). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Involvement of TRPV4 channels at the acupoints in the AP analgesic mechanism (n = 6–9, mean ± SE). (a,b) Effects of HC067047 (0.2 mM, 20 μL) injection in ST36 on AP-relieved PWT and PWL, respectively. Data illustrate the final behavioral tests. Numbers above each column denote sample size (n). *** p < 0.001. (c) Immunofluorescence staining for TRPV4 in ST36. Both skeletal muscle and skin layers displayed a high TRPV4 expression (green). Nuclei were stained with DAPI (blue). Scale bar: 100 μm. (d) TRPV4 mRNA expression in muscle and skin layers excised from normal rats at ST36 (n = 4–5).

Figure 5

Effect of TRPV4 inhibition in injured plantar on CFA-induced pain hypersensation (n = 6–9, mean ± SE). (a,b) Effects of HC067047 (0.2 mM, 20 μL) pre-injection in the injured plantar on CFA-induced tactile allodynia and thermal hyperalgesia, respectively. Data illustrate the final behavioral tests. Numbers above each column denote sample size (n). ** p < 0.01, *** p < 0.001.

Figure 6

Involvement of eATP hydrolysis in the acupoints in the AP analgesic mechanism (mean ± SE). (a,b) Effects of ARL67156 (0.1 mM, 50 μL) and apyrase (10 U, 50 μL) on AP-relieved PWT and PWL, respectively (n = 6–8). Data illustrate the final behavioral tests. In a, data in the AP + ARL group were non-normally distributed, so the difference between the AP + ARL group and AP group was compared with a Mann-Whitney U test. ** p < 0.01, *** p < 0.001. Numbers above each column denote sample size (n). (c) Time-course of eATP changes during needling on CFA model rats in the absence and presence of ARL67156 (0.1 mM). Each sample contained 1 min microdialysate. These are representative traces from n = 6–7 recordings. (d) NTPDase1–3 mRNA expression in muscle and skin layers excised from normal rats at ST36 (n = 4).

Figure 7

Effect of TRPV4 channel inhibition at the acupoints on needling-induced eATP accumulation (n = 4–5, mean ± SE). (a) Time-course of eATP changes during AP in the absence and presence of HC067047 (0.2 mM, 20 μL). Each sample contained 5 min microdialysate. (b) Comparison of eATP peaks and the total amounts, respectively, in the absence and presence of HC067047 (0.2 mM, 20 μL). Data based on (a). eATP amounts were calculated for the area under the curve from 0 to 20 min. * p < 0.05.