P2X receptors expression and ATP-induced calcium response in mouse peritoneal mast cells
Upon for instance tissue stress, hypoxia or inflammation, ATP is released from cells into the extracellular environment leading to the increase in extracellular ATP. Our results also showed that ATP was significantly increased in inflammatory pain such as CFA (0.4913 ± 0.04 µmol/g vs 1.116 ± 0.09 µmol/g, *** p < 0.001, Supplementary Fig. S2). Because mast cells play important roles in the pathological process of pain, we speculate that P2X receptors on mast cells involved in this function.
First, to identify P2X receptors on mast cell, we explored the P2X expression in mouse peritoneal mast cells by RT-PCR screen. As shown in Fig. 1A, we found that mouse peritoneal mast cells expressed several different ionotropic P2X receptors including P2X1, P2X3, P2X4 and P2X7. P2X receptor is a non-selective cation channel, the most obvious permeability to Ca2+. Hence, we examined the calcium influx in mouse peritoneal mast cells induced by ATP. The results showed that there were transient increases of intracellular calcium in mast cells treated by different concentrations of ATP (Fig. 1B, C). The ratios of fluorescence intensity were varied with different ATP concentrations, and EC50 was about 6.5 µM (Fig. 1C). In addition to fluorescence intensity, the reaction durations also existed differences (Fig. 1B), which indicated that different concentrations of ATP could activate mast cells through different P2X ionotropic receptors.
To confirm that, we used special P2X channel antagonists. As shown in Fig. 1D-H, the calcium influx caused by different concentrations of ATP could be partially blocked by a non-selective P2 purinergic receptor antagonist PPADS (20 µM, pre-incubation for 5 minutes). In addition, calcium influx caused by 1 µM ATP was inhibited by P2X1 receptor antagonist NF449 (1 µM, pre-incubation for 5 minutes) (Fig. 1D). AF-353 (P2X3 receptor antagonist, 0.1 µM, pre-incubation for 5 minutes) could reduce the calcium influx caused by 10 µM ATP (Fig. 1E). And the transient increase of the intracellular calcium induced by 100 µM ATP was blocked by 5-BDBD (1 µM, pre-incubation for 5 minutes) (Fig. 1F), which is a specific P2X4 receptor antagonist. Furthermore, the specific P2X7 receptor antagonist AZ10606120 (1 µM, pre-incubation for 5 minutes) had the ability of reducing the calcium influx caused by high concentration ATP such as 1 mM and 5 mM (Fig. 1G, H). These results indicated that P2X1, P2X3, P2X4 and P2X7 might contribute to the activation of mouse peritoneal mast cells.
Electrophysiological characteristics of mouse peritoneal mast cells induced by extracellular ATP
According to previously published literature, human mast cells are sensitive to ATP in a concentration-dependent manner [17]. Our experimental results proved that mouse peritoneal mast cells had the same characteristics. About 85% of mast cells were sensitive to 1 µM ATP (Fig. 2A, n = 17). The inward current could also be induced by 100 µM ATP (Fig. 2B, n = 21). When we increased the concentration of extracellular ATP to a high level, we found that both 1mM and 5 mM ATP had the ability to induce the inward currents repeatedly (Fig. 2C, n = 9; Fig. 2D, n = 8). As Fig. 2E, F shown, the current characteristics evoked by various concentrations of extracellular ATP were different from each other, including the amplitude of the inward current and the current durations. Although the current amplitude as well as the duration were different between 1mM ATP and 5 mM ATP, the inward currents had some similar characteristics such as “run-up” tendency (Fig. 2G, H). The current growth rate of second ATP application had no difference as Fig. 2I shown. The current growth rate is defined as. Furthermore, the inward current evoked by 1mM ATP was voltage-dependent (Fig. 2J-L). The activate curves and inactivate curves induced by 5 mM ATP were shown in Fig. 2M. According these curves, the conductance curve was calculated as shown in Fig. 2N, which indicated that the current induced by 5 mM ATP had characteristics with faster activation and slower inactivation.
Inward currents induced by extracellular ATP with different concentrations could be blocked by P2X1, P2X3, P2X4 and P2X7 antagonist
Electrophysiological results showed that there were different currents corresponding to ATP with different concentrations. Therefore, we speculated that several P2X receptor subtypes contributed to the activation progress. As Wareham described, P2X1,P2X4 and P2X7 receptors in LAD2 were activated by 1 µM ATP, 100 µM ATP and high concentrations of ATP, respectively [17]. In our study, we also found that 1 µM ATP hardly induced inward current when 20 µM PPADS (a non-selective P2 antagonist) (Fig. 3A, G, n = 9) or 1µM NF449 (the blocker of P2X1 receptor) (Fig. 3A, G, n = 13) was applied, which indicated that P2X1 was activated by 1 µM ATP. As Fig. 3B, H shown, 20 µM PPADS (n = 9) or 0.1 µM AF-353 (the blocker of P2X3 receptor, n = 10) could reduce the current evoked by 10 µM ATP, which indicated that P2X3 was activated by 10 µM ATP. In addition, P2X4 was involved in the current induced by 100 µM ATP, which was blocked by 1 µM 5-BDBD (Fig. 3C, I, n = 10) or 20 µM PPADS (Fig. 3C, I, n = 13). The current caused by 1mM ATP were inhibited by using 1 µM AZ 10606120 (the blocker of P2X7 receptor) (Fig. 3D, J, n = 5). The current induced by 5 mM ATP could also be inhibited by AZ 10606120 (Fig. 3E, K, n = 6). These results demonstrated that P2X7 receptor was activated by high concentrations of ATP such as 1 mM and 5 mM. Intrestingly, we found that 20 µM PPADS could partially inhibit the current induced by 1 mM ATP (Supplementary Fig. S3A, S3C), but had no effect on the current evoked by 5 mM ATP (Supplementary Fig. S3B, S3D), which indicated that PPADS might have limited inhibitory effect on P2X7. Furthermore, P2X7 receptor was sensitive to divalent cation as North described [24]. Consequently, our results illustrated that the inward current induced by 5mM ATP in the low divalent cation was greater than that in normal external solution (Fig. 3F, L), which confirmed that the existence of P2X7 in mouse peritoneal mast cells. Taken together, results confirmed that P2X1, P2X3, P2X4 and P2X7 expressed in mouse peritoneal mast cells and involved in the progress activation induced by extracellular ATP.
Activation of P2X7 receptor on mouse-derived mast cell could lead to degranulation and de novo synthesis of cytokines
Degranulation is one of important indicators of mast cell activation. Consistent with the research by Wareham et al in human mast cell line LAD2 [18], our results indicated that there is no detectable histamine release at lower concentrations of ATP such as 1 µM ATP and 100 µM ATP. However, histamine released from mouse peritoneal mast cells was significantly increased at higher concentrations of ATP (Fig. 4A). Besides histamine, mast cells are effective producers of inflammatory cytokines in response to various stimuli. According to previous literatures, cytokines secretion especially IL-1β induced by the extracellular ATP has been widely studied in different immune cells [8–12]. However, the release of cytokines from mast cells induced by extracellular ATP remains unclear. To examine the potential mediator release in mast cells, we detected a serious of mediators such as IL-6, IL-1β, CCL2 and CCL3. Due to the mouse peritoneal mast cells are too few to detect, we turned to mouse mastocytoma cells P815, which also expressed P2X1, P2X3, P2X4 and P2X7 receptors (Supplementary Fig. S4). Data in Fig. 4B-H demonstrated that the regulation of inflammatory mediators was related to the concentration as well. There was no significant change or up-regulated slightly in the expression of cytokines when treated with low concentration of ATP (Fig. 4B-D). ATP with high concentrations could significantly up-regulate the expression of a variety of inflammatory cytokines, such as IL-1β and CCL3 (Fig. 4E, G). In addition, we found that cytokines were regulated slightly after treatment with 5mM ATP for 4 hours, but IL-1β and CCL3 were significantly up-regulated after treatment for 0.5 hours, which might be related with the negative feedback caused by high ATP concentration (Fig. 4F, G). AZ10606120, a specific P2X7 receptor antagonist (5 µM, pre-incubation for 5 minutes), almost completely inhibited the upregulation of IL-1β and CCL3 induced by 1mM ATP (Fig. 4H). Therefore, we concluded that P2X7 receptor on mast cells could mediate mast cell degranulation and de novo synthesis of inflammatory factors such as IL-1β and CCL3.
High concentration of ATP could induce peripheral pain in mice by activating P2X7 channel on mast cells
Our experimental results indicated that P2X7 receptor on mast cell had the function of releasing inflammatory mediators, which might contribute to pain via neuro-immune interactions. We assumed that mast cells and P2X7 receptor promoted the peripheral pain induced by high concentration of ATP. In order to prove this hypothesis, we utilized the mast cell-deficient Kit (W-sh) Sash mutant mice and P2X7 receptor antagonist. Firstly, results showed that high concentration of ATP (100 mM, 20 µL, iH) did induce paw swelling (Fig. 5A, C), inflammatory cells infiltration (Fig. 5B), mast cells degranulation (Fig. 5D) and mechanical hyperalgesia (Fig. 5E). As our expected, mast cell-deficient mice could alleviate ATP-induced pain including paw swelling, mechanical withdrawal threshold and the infiltration of inflammatory cells (Fig. 5A-E). To further explore the mechanism, we also tested the inflammatory mediators. The RT-PCR data showed that the mRNA expression levels of IL-6, IL-1β, CCL2 and CCL3 were significantly upregulated for the C57/BL mice. However, only IL-6 and CCL3 were slightly upregulated for the Sash mice. In comparison with the C57/BL mice, the degree of upregulation of IL-1β, CCL2 and CCL3 for the Sash mice was reduced (Fig. 5F). At the same time, we also studied the function of P2X7 receptor in high concentration of ATP-induced peripheral pain. Our experimental results showed that AZ10606120 (specific P2X7 receptor antagonist, 2 mg/kg, ip, pre-administration 1h) could significantly reduce the pain behavior and paw thickness at 4h after ATP treatment (Fig. 5G, H).
P2X7 on mast cells is a potential target for salicylic acid and aspirin analgesia
P2X7 is an appealing target for anti-inflammatory therapy, so we used P2X7 as an analgesic target to screen several anti-inflammatory substances monomers. We found that Matrine, Higenamine, Dictamnine, Prim-O-glucosylcimifugin, Liquiritin, Menthol, Ferulic Acid, 3-Hydroxy-4-methoxycinnamic acid, Isoginkgetin, Vanillic acid, Luteolin, Isoliquiritigenin or Aloeemodin had no inhibitory effect on the current evoked by high concentration of ATP (Supplementary Table 1). Interestingly, salicylic acid and aspirin could inhibit the inward current generated by high concentration of ATP as Fig. 6A shown. Compared with the current amplitude induced by first ATP application, 300 µM, 500 µM or 1 mM salicylic acid could slightly inhibit the current amplitude induced by second ATP application (Fig. 6B-D, n = 21, n = 14 and n = 16 respectively). It is worth noting that the current induced by 5 mM ATP had a “run-up” tendency, which indicated that the current growth rate should be studied. Data showed that the current growth rate of second ATP application was significantly inhibited by salicylic acid as Fig. 6E shown. 500 µM or 1 mM aspirin could also inhibit the current amplitude induced by 5mM ATP (Fig. 6G-H, n = 6, n = 8 and n = 20 respectively). The current growth rate was significantly inhibited by 500 µM or 1 mM aspirin (Fig. 6I). The intercellular Ca2+ concentration assay results also showed that 300 µM salicylic acid or 1 mM aspirin could also inhibit 5mM ATP-induced calcium influx (Supplementary Fig. S5). At the same time, we also explored the effects of drugs on inflammatory mediators. As our expected, 300 µM salicylic acid or 1 mM aspirin could attenuate the up-regulation of IL-1β and CCL3 mediated by high concentration of ATP, especially IL-1β (Fig. 6J). Results from behavioral test indicated that salicylic acid (50 mg/kg, ig) or aspirin (50 mg/kg, ig) could also alleviate the peripheral pain induced by high concentration of ATP (Fig. 6K). To further clarify the relationship between salicylates and P2X7 receptor, we used the P2X7 receptor agonist BzATP. Results showed that 300 µM salicylic acid (n = 18) or 300 µM aspirin (n = 7) significantly inhibited the current growth rate (Fig. 6L) and the calcium influx (Supplementary Fig. S6) evoked by BzATP. These experimental results suggested that P2X7 on mast cells might be a potential target for salicylic acid and aspirin analgesia.
GDP binding region is the critical for the combination of salicylic acid and aspirin with P2X7
Our experimental results have shown that the analgesic effect of salicylic acid and aspirin may be achieved by inhibiting P2X7 channel. Next, we want to know how salicylic acid and aspirin work in combination with P2X7. To uncover this mystery, molecular docking was used to analyze the interaction between salicylic acid or aspirin and P2X7 by Discovery Studio software. As shown in Fig. 7, salicylic acid (Fig. 7G-L) and its derivative aspirin (Fig. 7A-F) had affinity to the GDP-binding region of P2X7, including A: GDP 703, B: GDP703 and C: GDP704 ligands. Among these, aspirin has the highest affinity with A: GDP 703 ligand as Fig. 7A, B shown. The -COOH of aspirin formed two Electrostatic-bonds with Arg546 (R546) and Arg578 (R578) of A: GDP 703 ligand, and van der Waals-bonds with Ser589 (S589). Consistent with the electrophysiological and the intercellular Ca2+ concentration assay results, we considered that salicylic acid and aspirin could inhibit P2X7 activation by directly binding the receptor.