NTPDase1-ATP-P2Y2Rs axis in the sciatic nerve contributes to acupuncture at "Zusanli" (ST36)-induced analgesia in ankle arthritis rats.

BACKGROUND: The efficacy of acupuncture at Zusanli (ST36) in alleviating lower-limb pain is widely acknowledged in clinical practice, while its underlying mechanism remains incompletely elucidated. Our previous research had revealed that the prompt analgesia induced by needling-ST36 was accompanied by expression alterations in certain exco-nucleotidases within the sciatic nerve. Building upon this finding, the current work focused on NTPDase1, the primary ecto-nucleotidase in the human body, which converts ATP into AMP. METHODS: A 20-min acupuncture was administered unilaterally at the ST36 on rats with acute ankle arthritis. The pain thresholds of the injured hind paws were determined. Pharmacological interference was carried out by introducing the corresponding reagents to the sciatic nerve. ATP levels around the excised nerve were measured using a luciferase-luciferin assay. Live calcium imaging, utilizing the Fura 2-related-F340/F380 ratio, was conducted on Schwann cells in excised nerves and cultured rat SCs line, RSC96 cells. RESULTS: The analgesic effect induced by needling-ST36 was impaired when preventing ATP degradation via inhibiting NTPDase1 activities with ARL67156 or Ticlopidine. Conversely, increasing NTPDase1 activities with Apyrase duplicated the acupuncture effect. Similarly, preventing the conversion of AMP to adenosine via suppression of NT5E with AMP-CP hindered the acupuncture effect. Unexpectedly, impeded ATP hydrolysis ability and diminished NTPDase1 expression were observed in the treated group. Agonism at P2Y2Rs with ATP, UTP, or INS365 resulted in anti-nociception. Contrarily, antagonism at P2Y2Rs with Suramin or AR-C 118925xx prevented acupuncture analgesia. Immunofluorescent labeling demonstrated that the treated rats expressed more P2Y2Rs that were predominant in Schwann cells. Suppression of Schwann cells by inhibiting ErbB receptors also prevented acupuncture analgesia. Finally, living imaging on the excised nerves or RSC96 cells showed that agonism at P2Y2Rs indeed led to [Ca2+]i rise. CONCLUSION: These findings strongly suggest that the analgesic mechanism of needling-ST36 on the hypersensation in the lower limb partially relies on NTPDase1 activities in the sciatic nerve. In addition to facilitating adenosine signaling in conjunction with NT5E, most importantly, NTPDase1 may provide an appropriate low-level ATP milieu for the activation of P2Y2R in the sciatic nerve, particularly in Schwann cells.

Mast Cell-Associated Serotonin in Acupoint Contributes to Acupuncture Analgesia in Arthritis Rats by Mediating ATP Release.

BACKGROUND: The activation of subcutaneous mast cells (MCs) helps to trigger the analgesic effect induced by acupuncture (AP), a traditional oriental therapy, that has been gradually accepted worldwide. This work aimed to reveal whether the serotonin (5-hydroxytryptamine, 5-HT) released from MCs plays an important role in this process, which has a controversial effect in the mechanism of pain. METHODS: In vivo tests, a 20-min session of AP was applied at Zusanli acupuncture point (acupoint) of acute ankle arthritis rats. Pain thresholds of the injured hindpaw were assessed to reflect the pain state, and the targeting substances in the interstitial space of the treated acupoint were sampled by microdialysis. In vitro experiments, exogenous 5-HT (exo-5-HT) was introduced to mediate adenosine triphosphate (ATP) release from cultured MCs. RESULTS: Needling promoted 5-HT accumulation at the Zusanli acupoint, which was prevented by sodium cromolyn. AP's analgesic effect was suppressed by the inhibition of 5-HT receptors at the acupoint, especially 5-HT1A subtype. In vitro tests, mechanical perturbation mimicking needling stimulation induced MCs to release 5-HT. 1 µM and 10 µM of exo-5-HT facilitated ATP release, which was restrained by blocking of 5-HT1 receptors rather than 5-HT3 receptors. As 5-HT, ATP and adenosine were also transiently accumulated in the treated acupoint during needling. Promoting ATP hydrolysis or activation adenosine A1 receptors duplicated AP analgesic effect. Finally, the inhibition of ATP receptors by suramin or pyridoxal phosphate-6-azo tetrasodium salt hydrate (PPADS) prevented AP analgesic effect. CONCLUSIONS: Our results suggest that MC-associated 5-HT release at acupoints contributes to AP analgesia, and the mediation of ATP secretion through 5-HT1A receptors might be the underlying mechanism at play. ATP could facilitate adenosine production or the propagation of needling signals.

The real-time detection of acupuncture-induced extracellular ATP mobilization in acupoints and exploration of its role in acupuncture analgesia.

Our and in vitro studies had confirmed that mechanosensitive ATP release and accumulation in acupoints was elicited by acupuncture (AP), which might be a pivotal step for triggering AP analgesia. But to date, the dynamics of extracellular ATP (eATP) in the interstitial space during AP process was poorly known, mainly due to the low temporal resolution of the current detection approach. This study attempted to capture rapid eATP signals in vivo in the process of needling, and further explored the role of this eATP mobilization in initiating AP analgesic effect. Ipsilateral 20-min needling was applied on Zusanli acupoint (ST36) of complete Freund's adjuvant (CFA)-induced ankle arthritis rats. Pain thresholds were assessed in injured-side hindpaws. eATP in the interstitial space was microdialyzed and real-time quantified by luciferin-luciferase assay at 1-min interval with the aid of the microfluid chip. We revealed in behavioral tests that modulation of eATP levels in ST36 influenced AP analgesic effect on ankle arthritis. A transient eATP accumulation was induced by needling that started to mobilize at 4 min, climbed to the peak of 11.21 nM within 3.25 min and gradually recovered. Such AP-induced eATP mobilization was significantly impacted by ankle inflammation, needling depth, needle manipulation, and the presence of local ecto-nucleotidases. This work reveals that needling elicits a transient eATP mobilization in acupoints, which contributes to initiating AP analgesia. This study will help us better understand the peripheral mechanism of AP analgesia and guide clinicians to optimize the needle manipulations to improve AP efficacy.

Activation of Subcutaneous Mast Cells in Acupuncture Points Triggers Analgesia.

This review summarizes experimental evidence indicating that subcutaneous mast cells are involved in the trigger mechanism of analgesia induced by acupuncture, a traditional oriental therapy, which has gradually become accepted worldwide. The results are essentially based on work from our laboratories. Skin mast cells are present at a high density in acupuncture points where fine needles are inserted and manipulated during acupuncture intervention. Mast cells are sensitive to mechanical stimulation because they express multiple types of mechanosensitive channels, including TRPV1, TRPV2, TRPV4, receptors and chloride channels. Acupuncture manipulation generates force and torque that indirectly activate the mast cells via the collagen network. Subsequently, various mediators, for example, histamine, serotonin, adenosine triphosphate and adenosine, are released from activated mast cells to the interstitial space; they or their downstream products activate the corresponding receptors situated at local nerve terminals of sensory neurons in peripheral ganglia. The analgesic effects are thought to be generated via the reduced electrical activities of the primary sensory neurons. Alternatively, these neurons project such signals to pain-relevant regions in spinal cord and/or higher centers of the brain.