A1 adenosine receptor activation inhibits P2X3 receptor-mediated ATP currents in rat dorsal root ganglion neurons

doi:10.21203/rs.3.rs-1613085/v1

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A1 adenosine receptor activation inhibits P2X3 receptor-mediated ATP currents in rat dorsal root ganglion neurons

https://doi.org/10.21203/rs.3.rs-1613085/v1

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Purinergic signaling plays an important role in multiple pain processes. P2X3 receptor is considered to be a target in pain therapeutics, and A1 adenosine receptor signaling is essential for analgesia. However, it is still unclear whether there is a link between A1 adenosine receptors and P2X3 receptors in pain process. Herein, we show that the A1 adenosine receptor agonist N⁶-cyclopentyladenosine (CPA) suppressed α,β-methylene-ATP (α,β-meATP)-evoked inward currents mediated by P2X3 receptors in a concentration- dependent and voltage-independent manner in rat dorsal root ganglion (DRG) neurons. CPA shifted the concentration-response curve for α,β-meATP downwards, with a decrease of 38.44 ± 5.87% in the maximal current response of P2X3 receptors to α,β-meATP in the presence of CPA. The CPA-induced suppression was blocked by the A1 adenosine receptor antagonist KW-3902 and prevented by intracellular application of either the G_i/o-protein inhibitor pertussis toxin, the adenylate cyclase activator forskolin, or the cAMP analog 8-Br-cAMP. CPA also suppressed the membrane excitability of DRG neurons, with a significant decrease in the amplitude of the depolarization and the number of spikes induced by α,β-meATP in the presence of CPA. Finally, intraplantar pretreatment of CPA dose-dependently relieved α,β-meATP-induced nociceptive responses in rats through peripheral A1 adenosine receptors. These results suggested that CPA suppressed the functional activity of P2X3 receptors in rat primary sensory neurons via A1 adenosine receptor, intracellular G_i/o-proteins and cAMP signaling cascades, suggesting a novel peripheral mechanism underlying its analgesic effect.

A1 adenosine receptor

P2X3 receptor

electrophysiology

dorsal root ganglion neuron

nociceptive behavior.

Purines, including ATP, ADP and adenosine and their receptors are deeply involved in the pathophysiology of pain [1]. Among of all purine subtypes, adenosine/A1 adenosine receptor signaling has been shown to be essential for analgesia [2, 3]. Adenosine can exert an analgesic effect on pain behaviours in different models of acute and chronic pain by activating A1 adenosine receptors [3, 4]. A1 adenosine receptor is widely distributed in peripheral sensory nerve endings, dorsal horn of spinal cord and supraspinal pain-processing structures [3, 5]. Studies have shown that activation of A1 adenosine receptors generates unambiguous analgesic action in models of postoperative, visceral, neuropathic and inflammatory pain [6–10]. Acupuncture and various natural compounds induce analgesia via a local increase in adenosine and/or activation of A1 adenosine receptors [11–16]. Positive allosteric modulators of A1 adenosine receptors have also efficacy in reducing pain [17, 18]. On the contrary, it has been shown that downregulation of adenosine and A1 adenosine receptors contributes to neuropathic pain [19]. Animals that lacking the A1 adenosine receptor exhibit increased nociceptive responses to noxious stimuli [20, 21]. Peripheral A1 adenosine receptor contributes to the analgesic effect of adenosine signaling. Intraplantar injection of adenosine and other A1 adenosine receptor agonists produces antinociceptive responses [22, 23]. Recently, intraplantar administration of adenosine A1 receptor agonist has been reported to induce an anti-nociceptive effect on glutamate-induced nociceptive behavior [24]. Locally administration of selective A1 adenosine receptor antagonists can reverse the analgesic effects of acetaminophen and tramadol, indicating the contribution of peripheral A1 adenosine receptors to analgesia [25, 26]. Although previous studies show the contribution of peripheral A1 adenosine receptor to antinociception, the exact mechanisms underlying analgesia remain to be defined.

Apart from A1 adenosine receptors, other purinergic receptors such as P2X3 receptors are also expressed in nociceptive afferent pathway, including in terminals of nociceptive fibers [27, 28]. P2X3 receptor has been shown to participate in multiple pain processes [29, 30]. Antagonists, antisense oligonucleotide and siRNA of P2X3 receptors are effective in pain relief [31–34]. Mice lacking P2X3 receptors display attenuated spontaneous pain behaviors to administration of ATP or in formalin test [35, 36]. Since multiple purinergic signals have been shown to play roles in pain, is there a functional interaction between them in pain process? Previous studies have shown that activation of metabotropic P2Y receptors can inhibit ionotropic P2X3 receptors [37–39]. However, it was still unclear whether P2X3 receptor can be modulated by the activation of A1 adenosine receptors. The present study reports that the A1 adenosine receptor agonist N⁶-cyclopentyladenosine (CPA) did not only inhibit the electrophysiological activity mediated by P2X3 receptors in rat DRG neurons via an intracellular cAMP pathway, but also relieved P2X3 receptor-mediated nociceptive behaviors in rats by activating peripheral A1 adenosine receptors.

2.1 Preparation of DRG neurons

All experimental protocols were approved by the Animal Research Ethics Committee of Hubei University of Science and Technology (2016-03-005). Sprague-Dawley rats (5–6 weeks old) were anesthetized and sacrificed. The DRGs from the rats were removed and chopped. The minced ganglia were transferred to a test tube containing Dulbecco's modified Eagle's medium (DMEM) and incubated with shaking for 25–30 min at 35°C. The incubation solution contained 1.0 mg/ml of collagenase, 0.5 mg/ml of trypsin and 0.1 mg/ml of IV DNase. Trypsin digestion was terminated by adding1.25 mg/ml of soybean trypsin inhibitor. The cells were cultured for 12–24 h at 37°C in DMEM containing nerve growth factor (100 ng/ml) and fetal bovine serum (10%).

2.2 Electrophysiological recordings

Electrophysiological experiments were performed as described previously [40]. An EPC-10 patch clamp amplifier (HEKA Electronic, Lambrecht, Germany) were used for the whole-cell patch clamp recordings. The isolated DRG neurons weretransferred to a 35- mm culture dish and kept in normal external solution for at least 60 min before electrophysiological recordings. The external solution contained the following (in mM): 150 NaCl, 5 KCl, 2 MgCl₂, 2.5 CaCl₂, 10 HEPES, and 10 D-glucose. The pH and osmolarity were adjusted to 7.4 with NaOH and 330 mOsm/L with sucrose, respectively. The recording pipettes were pulled using a Sutter P-97 puller (Sutter Instruments, CA, USA), whose resistance was in the range of 3–6MΩ. The micropipette solution contained the following (in mM): 140 KCl, 2 MgCl₂, 11 EGTA, 10 HEPES, 4 ATP, and 0.3Na₂GTP. The pH and osmolarity were adjusted to 7.2 with KOH and 310 mOsm/L with sucrose, respectively. After whole-cell configuration was established, 70–80% series resistance and membrane capacitance current were compensated. The recorded currents were sampled at 10 kHz and filtered at 2 kHz. Only small- and medium-sized nociceptive DRG neurons (15–40µm in diameter) were used for the electrophysiological recordings. The membrane potential of the neurons was clamped at -60 mV. Only DRG neurons with a resting membrane potential less than − 50 mV were used for current-clamp recordings.

2.3 Drug application

All drugs were obtained from Sigma-Aldrich (St. Louis, MO, USA). The working concentration of drugs was freshly prepared in normal external solution. Each working drug was stored in a series of independent reservoirs and subjected to gravity. The distance between the drug exit and the recorded neurons was approximately 30 µm. To block G-protein and intracellular signaling, some antagonists or blockers were dissolved in the internal solution and applied for intracellular dialysis through recording patch pipettes as described previously [40]. To ensure that dialysis drugs are infused into the cell interior, current recording was performed at least 30 min after cell membrane rupture.

2.4 Nociceptive behavior induced by α,β-meATP in rats

Rats were first habituated for 30 minutes in a Plexiglas chamber during the nociceptive behavioral experiment. The rats in six different groups received intraplantar injections of 50µl vehicle, different doses (0.1, 1 and 10ng ) of CPA, 30ng KW-3902 + 10ng CPA, respectively. After 10 min, another experimenter injected α,β-methylene-ATP (α,β-meATP, 50µg in 50µl) into the ipsilateral hind paw and tested the nociceptive behavior. The assessor of the behavioral measures was blinded to the prior treatment conditions. Nociceptive behaviors (that is, number of flinches) were monitored over a 10 min period starting after the injection of α,β-meATP.

2.5 Data analysis

All data were expressed as mean ± S.E.M,and statistically compared using Student's t-test or analysis of variance (ANOVA), followed by Bonferroni’s post hoc test. The nonlinear curve-fitting program ALLFIT was used for statistical analysis of the concentration–response data.

3.1 CPA concentration-dependently inhibits P2X3 receptor-mediated ATP currents in rat DRG neurons

In the majority of tested DRG cells (76.5%, 13/17), an application of 100 µM α,β-methylene-ATP (α,β-meATP) or 100 µM ATP for 5 sec evoked a rapid inward currents (I_ATP) under holding potentials of − 60 mV conditions (Fig. 1A). The I_ATP was completely blocked by the specifical P2X3 or P2X2/3 receptor antagonist A-317491(100 µM), but not by the P2X4 receptor antagonist PSB-12062 (10µM) and the potent P2X7 receptor antagonist A438079 (1µM), indicating that the I_ATP was mediated by P2X3 or P2X2/3 receptors.

In most DRG neurons (69.2%, 9/13), we observed that the peak amplitude of 100 µM α,β-meATP- and ATP-activated currents decreased when 100 nM CPA was pre-treated to DRG cells for 5min prior to the next recording (Fig. 1B). The decrease of the I_ATP amplitude depended on the concentration of pre-treated CPA. The sequential current traces in Fig. 1C illustrate that a gradual decrease in the amplitude of the currents evoked by 100 µM α,β-meATP with an increase in CPA concentration from 3 nM to 300 nM in a representative DRG cell. Figure 1D shows the concentration-effect curve of CPA on 100 µM α,β-meATP evoked I_ATP with an IC₅₀ (half-maximal effective concentration) value of 39.40 ± 3.18 nM. These results suggest that CPA inhibited P2X3 receptor-mediated ATP currents in a concentration-dependent manner.

3.2 Concentration–response and current–voltage relationships for α,β-meATP in the absence and presence of CPA

We studied whether the suppression of CPA depends on the concentration of α,β-meATP. Figure 2A shows that pre-application of CPA (100 nM for 5 min) decreased the amplitudes of ATP currents evoked by 3, 30, and 300 µM α,β-meATP, respectively. The concentration-response curves in Fig. 2B were plotted using a series of concentration of α,β-meATP in the absence and presence of 100 nM CPA, which were fit with the Hill equation. The concentration-response curve for α,β-meATP in the presence of 100 nM CPA was shifted downwards with a decrease of 38.44 ± 5.87% in maximal current response, which was evoked by 300 µM α,β-meATP (P < 0.01, Bonferroni’s post hoc test). However, the Hill coefficient or the slope were not significantly different between the two curves, which were 1.36 ± 0.31 and 1.28 ± 0.26 in the absence and presence of CPA, respectively (P > 0.1, Bonferroni’s post hoc test). In addition, CPA pre-treatment did also not change the EC₅₀ of α,β-meATP for P2X3 receptors, which were 29.23 ± 3.06 µM and 27.42 ± 2.94 µM in the absence and presence of CPA, respectively (P > 0.1, Bonferroni’s post hoc test). These results suggest that CPA inhibited the maximum response to α,β-meATP, but not shifted the sensitivity of P2X3 receptors to α,β-meATP.

We then investigated whether the suppression of ATP currents by CPA depends on clamping potentials. Figure 2C shows that pre-application of CPA (100 nM for 5 min) decreased the amplitudes of ATP currents evoked by 30 µM α,β-meATP when the membrane potential was clamped at − 80 mV, − 40 mV and + 20 mV, respectively. Figure 2D shows that the current–voltage (I–V) curves for α,β-meATP in the absence and presence of 100 nM CPA, which were plotted under a series of clamping potentials conditions. CPA pre-treatment decreased the slope of I–V curve with no significant difference in the CPA-induced suppression on ATP currents at different clamping potentials from − 80 to 20 mV (P > 0.1, Bonferroni’s post hoc test). CPA did not change the reversal potential (near 0 mV). The results indicate that CPA suppressed P2X3 receptor-mediated ATP currents in a voltage-independent manner.

3.3 Participation of A1 adenosine receptors, G _i/o proteins and cAMP signaling in the CPA-induced inhibition of ATP currents

Consider that CPA is a selective A1 adenosine receptor agonist, we identified whether A1 adenosine receptors mediate the CPA-induced suppression of α,β-meATP-evoked currents. KW-3902, an A1 adenosine receptor antagonist, was co-applied with CPA to DRG neurons. Figure 3A and B show that the CPA-induced suppression of I_ATP was significantly blocked by KW-3902. The amplitude of I_ATP decreased from 1.36 ± 0.13 nA to 0.79 ± 0.09 nA in 8 DRG cells pre-treated with 100nM CPA alone for 5 min (P < 0.01, one-way ANOVA followed by post hoc Bonferroni’s test). In contrast, the amplitude of I_ATP was 1.22 ± 0.11 nA in 8 DRG neurons co-treated with 300 nM KW-3902 and 100 nM CPA, which was significantly different from the I_ATP amplitude after CPA pre-treatment alone (P < 0.01, one-way ANOVA followed by post hoc Bonferroni’s test, n = 8). These results indicate that CPA inhibited ATP currents in DRG neurons through A1 adenosine receptors.

A1 adenosine receptor belongs to G_i/o protein-coupled receptor family, which can trigger a cascade of intracellular events. We then identified which known signaling pathways downstream of A1 adenosine receptor were involved in the suppression of ATP current by CPA. Firstly, we explored whether G_i/o proteins contribute to the CPA-induced suppression of ASIC currents. A G_i/o protein inhibitor, pertussis toxin (PTX, 1 µg/ml), was applied internally to recorded DRG neurons before CPA treatment. We observed that CPA-induced suppression of I_ATP amplitude was significantly prevented by PTX (Fig. 3C and D). Secondly, we further explore intracellular signal transduction mechanisms underlying the CPA-induced suppression. The adenylate cyclase activator forskolin or the cAMP analog 8-Br-cAMP were applied internally to DRG cells through recording patch pipettes. Unlike under the normal internal solution conditions, pre-application of CPA (100 nM for 5 min) produced a decrease of 41.78 ± 2.76% on I_ATP. After forskolin (0.1 µM) or 8-Br-cAMP (1 mM) was applied internally to DRG cells, CPA caused only decreases of 3.04 ± 2.92% and 9.59 ± 1.79% on I_ATP, respectively (P < 0.01, compared with normal internal solution, one-way ANOVA followed by post hoc Bonferroni’s test, n = 8; Fig. 3C and D). These data collectively indicate that the suppression of ATP currents by CPA depended upon G_i/o proteins and downstream cAMP signaling pathway.

3.4 CPA-induced suppression of α,β-meATP-evoked membrane excitability of rat DRG neurons

We further investigated the effect of CPA on membrane excitability triggered by α,β-meATP. In the same DRG cell, 10 µM α,β-meATP did not only induce a large inward current under voltage-clamp conditions, but also evoked burst of action potentials (APs) under current-clamp conditions (Fig. 4A and B). Consistent with the results under voltage-clamp conditions, pre-application of CPA (100 nM for 5 min) decreased the number of α,β-meATP-evoked APs from 4.83 ± 0.60 of control condition to 2.17 ± 0.48 of CPA pre-treatment in 6 recorded DRG neurons (P < 0.01, paired t-test, n = 6; Fig. 4B).

We then observed the membrane potential of DRG neurons in the presence of TTX (1 µM) to block Na⁺ channel-mediated APs. As shown in Fig. 4C, pre-application of CPA (100 nM for 5 min) also decreased the depolarization evoked by 100µM α,β-meATP. In 6 tested neurons, the magnitude of membrane potential depolarization (ΔVm) decreased from 24.04 ± 1.86 mV to 17.64 ± 1.63 mV after 100 nM CPA pre-treatment for 5 min (paired t-test, P < 0.01, n = 7; Fig. 4D). These results indicate that CPA suppressed α,β-meATP-induced membrane excitability of rat DRG neurons.

3.5 Relief of α,β-meATP-evoked nociceptive behaviorsby CPA in rats

Finally, we investigated whether the suppression of P2X3 receptors by CPA in vitro plays a role in the nociceptive behaviors induced by α,β-meATPin vivo. Figure 5A shows intraplantar injection of α,β-meATP (50µg in 50µl) caused an intense spontaneous flinching/shaking response in rats, which was relieved by intraplantar pre-treatment of CPA. Quantitative analysis showed that CPA dose-dependently (0.1, 1 and 10ng) decreased the number of flinches induced by α,β-meATP (p < 0.05 and 0.01, one-way ANOVA followed by post hoc Bonferroni’s test, n = 8 ). Figure 5 shows that the relief of α,β-meATP-induced nociceptive behaviors by 10 ng CPA was blocked by co-treated the A1 adenosine receptor antagonist KW-3902 (30 ng; p < 0.01, one-way ANOVA followed by post hoc Bonferroni’s test, n = 8). In addition, α,β-meATP-induced nociceptive behaviors were not affected if 10 ng CPA was injected into the contralateral paws (data not shown). The results suggest that CPA relieved the α,β-meATP-induced nociceptive behaviors in vivo through activating peripheral A1 adenosine receptors.

The present data demonstrated that the A1 adenosine receptor agonist CPA suppressed the functional activity of peripheral P2X3 receptors. CPA decreased the amplitude of ATP currents and the membrane excitability induced by α,β-meATP in rat DRG neurons, which was involved A1 adenosine receptors, PTX-sensitive Gi/o proteins and cAMP signaling cascades. Behaviorally, CPA also relieved α,β-meATP-induced nociceptive behaviors in rats through peripheral A1 adenosine receptors.

It has been reported that P2X3 homomeric and P2X2/3 heteromeric receptors are the most prevalent isoforms in sensory neurons, especially in a subset of small- and medium-sized nociceptive neurons [28, 27]. In the present experiments, α,β-meATP-induced ATP currents in small- and medium-sized DRG nociceptive neurons (15–40µm in diameter) were blocked by specific antagonist of P2X3 and P2X2/3 receptors, but not by antagonists of P2X4 receptors and P2X7 receptors. Moreover, α,β-meATP can only activate P2X3 and P2X1 receptors [41]. Therefore, these recorded ATP currents were identified as currents mediated by P2X3 receptors, at least by P2X3-containing receptors. The present study showed that CPA concentration-dependently suppressed the recorded ATP currents through A1 adenosine receptors, indicating P2X3 receptor was a downstream regulatory target of CPA and A1 adenosine receptor signaling. Consistent with current results, other ion channels are also regulated by activation of A1 adenosine receptors, which may be a potential mechanism underlying its anti-nociceptive effect. For example, the A1 adenosine receptor agonists modulate the function of GABA_A and glycine receptors in rat primary sensory and spinal cord neurons [42, 43]. Voltage-dependent Ca²⁺ channels and inward rectifier K⁺ channels are also inhibited by activation of A1 adenosine receptors [44, 45]. We observed that the CPA-induced inhibition did not alter the sensitivity of P2X3 receptor to α,β-meATP, but significantly decreased the maximum response to α,β-meATP. Activation of A1 adenosine receptors has been shown to reduce AMPA receptor surface expression through internalization [46]. It remains to be further studied whether CPA inhibited P2X3 receptors through a similar mechanism.

The CPA-induced suppression of ATP currents was completely blocked by the A1 adenosine receptor antagonist KW-3902, indicating the effect of CPA was dependent upon the activation of A1 adenosine receptors. Both A1 adenosine receptors and P2X3 receptors have been shown to be present in DRG neurons [19, 3, 5, 28, 27]. Although the evidence of morphological co-existence remains to be elucidated, it was possible that CPA decreased ATP currents in some DRG neurons, where A1 adenosine receptors and P2X3 receptors are co-locate in the same cells. On the contrary, CPA failed to change ATP currents in some DRG cells, one possible explanation is that the DRG neurons only express P2X3 receptors, but not A1 adenosine receptors.

The A1 adenosine receptor is coupled to G_i/o member of the G protein family, through which it can inhibit adenylyl cyclase activity and decrease intracellular cAMP levels [47–49]. The present data showed that CPA-induced inhibition of ATP currents was lack after DRG neurons were intracellularly dialyzed with the G_i/o protein inhibitor PTX, the adenylate cyclase activator forskolin, or the cAMP analog 8Br-cAMP, indicating involvement of G_i/o-proteins and intracellular cAMP signaling. Our recent study has shown that CPA suppresses acid sensing ion channels via A1 adenosine receptors and intracellular G_i/o-proteins and cAMP signaling cascades in rat DRG neurons [50]. Consistent with current results, previous studies have shown that ATP-induced currents are modulated by intracellular cAMP-PKA signaling [51, 52]. cannabinoids inhibit ATP-activated currents in rat trigeminal ganglionic neurons by activating CB1 receptors and inhibiting the adenylate cyclase-cAMP-PKA signaling pathway [53]. Recent studies have reported that blockage of HCN channels inhibits the function of P2X2 and P2X3 receptors in rat DRG neurons via the cAMP-PKA signaling pathway [54]. Leu-enkephalin inhibits P2X3 currents in DRG neurons through G_i/o-proteins, while it increases P2X3 currents through PLC signaling pathway after pre-treatment of the neurons with a G_i/o-protein inhibitor PTX [55]. The present and previous studies suggested P2X3 receptor was downstream target of PTX-sensitive G_i/o proteins and cAMP signaling cascades.

P2X3 receptor is a cation-permeable channel. Once activated, it evokes an inward current, which is sufficient to result in membrane potential depolarization and even burst of APs [56]. Under the current-clamp conditions, CPA also suppressed α,β-meATP-induced membrane excitability of rat DRG neurons, including APs and membrane potential depolarization. These two results corroborated each other in the current-clamp and voltage-clamp experiments. P2X3 receptors are expressed in peripheral nociceptive sensory nerve endings, along with the soma of DRG neurons [27, 28]. Injection of ATP into the skin elicits pain via P2X3 receptors [35]. Injection of α,β-meATP into a hind paw also evokes spontaneous nociceptive behaviors in rats, such as licking, biting and lifting of the injected paw, which significantly blocked by the P2X3 receptor antagonists, P2X3 receptor antisenses, and P2X3 gene deletion [33, 35, 31]. These data indicate that P2X3 receptor plays an important role in generating pain at peripheral nerve endings. Within the periphery, A1 adenosine receptors are also localized on sensory nerve endings [3, 5]. The present results showed that peripheral pre-treatment of the CPA dose-dependently relieved the α,β-meATP-triggered nociceptive behaviors. The effects of CPA occurred locally by directly activating peripheral A1 adenosine receptors, since the anti-nociceptive effect was completely blocked by intraplantar administration of the A1 adenosine receptor antagonist KW-3902. These behavioral findings apparently confirmed the aforementioned electrophysiological results that CPA suppressed ATP-evoked currents, membrane potential depolarization and bursts of APs in DRG neurons through A1 adenosine receptors.

Purinergic signaling play a well-established role in the processing of nociceptive sensory signals in different pain models. The same purine molecule can regulate pain by activating different purinoceptors, also through the interaction between activated purinoceptors. For example, ATP can activate ionotropic P2X3 and metabotropic P2Y2 receptors. Activation of P2Y receptors has been shown to reversibly inhibit inward currents mediated by P2X3 receptors in rat DRG neurons [37–39]. The present study showed that different purine molecules such as adenosine and ATP regulate pain not only by activating cognate A1 adenosine receptors and P2X3, respectively, but also through the interaction between the two different purinoceptors. Adenosine is mainly metabolized from ATP, also is a precursor for ATP synthesis in vivo. Under inflammatory conditions, adenosine levels can reach up to 100 µM [57, 48]. A functional crosstalk between A1 adenosine receptors and P2X3 would provide a homeostatic mechanism to prevent excessive ATP signaling through P2X3 receptors. Clinically, adenosine and its receptors represent a target for pharmacological treatment of pain. But A1 adenosine receptor is widely expressed not only in the central nervous system but in the heart and adipose tissue, its agonists may elicit dose-limiting side effects such as bradycardia [58]. The present results that suppression of P2X3 receptors by CPA in primary sensory neurons provided a novel peripheral mechanism for analgesics targeting peripheral A1 adenosine receptors. CPA can exert its analgesic effects by inhibiting periphery P2X3 receptors, indicating P2X3 receptor may be therapeutic target for peripheral A1 adenosine receptor analgesia.

In summary, our results suggested that CPA suppressed P2X3-mediated the electrophysiological activity and pain, revealing a novel mechanism underlying its analgesic effect. P2X3 receptor can be used as a target for the analgesia of the activation of peripheral A1 adenosine receptors.

Statement of conflicts of interest:

We have no conflict-of-interest to declare.

Ethics approval

The animal study was reviewed and approved by the Animal Research Ethics Committee of Hubei University of Science and Technology (2016-03-005).

Consent to participate

Not applicable.

Consent for publication

All authors consent to publication of this manuscript.

Availability of data and materials

All data generated during this study are included in this article or are available on reasonable request from the corresponding authors.

Competing interests

All authors declare no conflicts of interest.

Funding

This work was supported by the National Natural Science Foundation of China (No. 81671101).

Authors' contributions

WPH designed this research. JWH, WLQ, QL, SW, XML, TTL and CYQ performed the experiments. JWH, WLQ and QL participated in data analysis. JWH, and WPH wrote the paper. All authors contributed substantially to this research and reviewed this manuscript.

Acknowledgements

We are grateful to Miss. Ying Jin for providing the technical support to carry out this work.

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A1 adenosine receptor activation inhibits P2X3 receptor-mediated ATP currents in rat dorsal root ganglion neurons

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Abstract

Figures

1 Introduction

2 Materials And Methods

2.1 Preparation of DRG neurons

2.2 Electrophysiological recordings

2.3 Drug application

2.4 Nociceptive behavior induced by α,β-meATP in rats

2.5 Data analysis

3 Results

3.1 CPA concentration-dependently inhibits P2X3 receptor-mediated ATP currents in rat DRG neurons

3.2 Concentration–response and current–voltage relationships for α,β-meATP in the absence and presence of CPA

3.4 CPA-induced suppression of α,β-meATP-evoked membrane excitability of rat DRG neurons

3.5 Relief of α,β-meATP-evoked nociceptive behaviorsby CPA in rats

4 Discussion

5 Conclusions

Declarations

References

Supplementary Files

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