Suppression of P2X3 receptor‐mediated currents by the activation of α2A‐adrenergic receptors in rat dorsal root ganglion neurons

Abstract Aims The α2‐adrenergic receptor (α2‐AR) agonists have been shown to be effective in the treatment of various pain. For example, dexmedetomidine (DEX), a selective α2A‐AR agonist, can be used for peripheral analgesia. However, it is not yet fully elucidated for the precise molecular mechanisms. P2X3 receptor is a major receptor processing nociceptive information in primary sensory neurons. Herein, we show that a functional interaction of α2A‐ARs and P2X3 receptors in dorsal root ganglia (DRG) neurons could contribute to peripheral analgesia of DEX. Methods Electrophysiological recordings were carried out on rat DRG neurons, and nociceptive behavior was quantified in rats. Results The activation of α2A‐ARs by DEX suppressed P2X3 receptor‐mediated and α,β‐methylene‐ATP (α,β‐meATP)‐evoked inward currents in a concentration‐dependent and voltage‐independent manner. Pre‐application of DEX shifted the α,β‐meATP concentration‐response curve downwards, with a decrease of 50.43 ± 4.75% in the maximal current response of P2X3 receptors to α,β‐meATP in the presence of DEX. Suppression of α,β‐meATP‐evoked currents by DEX was blocked by the α2A‐AR antagonist BRL44408 and prevented by intracellular application of the Gi/o protein inhibitor pertussis toxin, the adenylate cyclase activator forskolin, and the cAMP analog 8‐Br‐cAMP. DEX also suppressed α,β‐meATP‐evoked action potentials through α2A‐ARs in rat DRG neurons. Finally, the activation of peripheral α2A‐ARs by DEX had an analgesic effect on the α,β‐meATP‐induced nociception. Conclusions These results suggested that activation of α2A‐ARs by DEX suppressed P2X3 receptor‐mediated electrophysiological and behavioral activity via a Gi/o proteins and cAMP signaling pathway, which was a novel potential mechanism underlying analgesia of peripheral α2A‐AR agonists.


| INTRODUC TI ON
Noradrenaline is a major monoaminergic neurotransmitter and has many important functions through action on adrenergic receptors, such as α 1 , α 2 , and β receptors. 1 It has known that noradrenaline is also involved in pain modulation. Among three adrenergic receptors, α 2 -adrenergic receptors (α 2 -ARs) play a key role in mediating pain modulatory effects of noradrenaline. 2,3 The α 2 -ARs are distributed in the pain signaling pathway, including primary afferents and spinal dorsal horn. [4][5][6][7] In the spinal cord, norepinephrine released from descending pathways results in a presynaptic inhibition of pain by activating α 2 -ARs on central terminals of primary afferent nociceptors. 2 The α 2 -AR agonists can also mimic the noradrenergic projection of descending pain inhibition. 2 Dexmedetomidine (DEX), a potent highly selective α 2A -AR agonist, has shown potential analgesic effects in animals and humans when administered intrathecally or systemically. [8][9][10] The α 2 adrenergic drugs that include DEX and clonidine are approved as analgesic agents in clinical settings. 11 Systemic DEX analgesia could be blocked by peripheral α 2 -AR antagonists in neuropathic pain, suggesting a peripheral antinociceptive effect of DEX. 12,13 The peripheral effect of DEX on nociception is mediated by peripheral α 2 -ARs, which have been identified in the dorsal root ganglion (DRG). Mechanisms of DEX peripheral analgesia may underlie the modulation of a number of ligand-gated and voltage-gated ion channels, which are also expressed in DRG neurons. For example, DEX has been found to inhibit sodium channels through α 2 -ARs located on DRG and the trigeminal ganglion neurons. 14,15 DEX also suppresses the activity of TRPV1 via an α 2 -ARs and cAMP / protein kinase A (PKA) signaling pathway in DRG neurons. 16 P2X3 receptor is a purinergic ATP receptor. P2X3 homomeric and P2X2/3 heteromer receptors are distributed in DRG neurons and mainly located in a subset of small and medium-sized nociceptive neurons. [17][18][19] These peripheral P2X3-containing receptors contribute to the transmission of nociceptive signaling. For example, blocking P2X3 receptors by antagonists or antisense oligonucleotide can effectively reduce nociception. 20,21 P2X3 receptor-mediated currents in DRG neurons and nociceptive behaviors increase after inflammation and nerve injury. [22][23][24][25] It has shown that P2X3 receptors are regulated by adrenergic signaling.
The mRNA assessments indicate both α 1 -ARs and α 2 -ARs are expressed in DRG. 3,4 Noradrenaline potentiates ATP-evoked currents in DRG neurons by activating PKC via G q protein-coupled α 1 -ARs. 26 Considering the presence of α 2 -ARs and P2X3 receptors in DRG neurons, it was still unclear whether P2X3 receptors were also modulated by activation of α 2 -ARs. Herein, we observed that the activation of α 2A -ARs by DEX inhibited the electrophysiological activity of P2X3 receptors via an intracellular cAMP signaling pathway in rat DRG neurons. DEX also relieved P2X3 receptormediated nociceptive behaviors in rats by activating peripheral α 2A -ARs.

| Preparation of DRG neurons
All experimental protocols were approved by the animal research ethics committee of Hubei University of Science and Technology.
All animal data reporting has followed the ARRIVE 2.0 guidelines (PMID: 32663096). Sprague-Dawley male rats (5-6 weeks old) were anesthetized and then killed. The DRGs from rats were removed and chopped. The minced ganglia were transferred to a test tube containing Dulbecco's modified Eagle's medium (DMEM) and incubated in a shaking for 25-30 min at 35°C. Incubation solution contained 1.0 mg/ml collagenase, 0.5 mg/ml trypsin, and 0.1 mg/mL IV DNase.
Trypsin digestion was terminated by adding 1.25 mg/mL soybean trypsin inhibitor. The cells were cultured for 12-24 hours at 37°C in DMEM containing never growth factor (100 ng/mL) and fetal bovine serum (10%).

| Electrophysiological recordings
Electrophysiological experiments were performed as described previously. 27,28 MultiClamp-700B amplifier and Digidata-1550B A/D converter (Axon Instruments, CA, USA) were used for wholecell patch clamp recordings. The isolated DRG neurons were transferred 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 MgCl 2 , 2.5 CaCl 2 , 10 HEPES, and 10 d-glucose. Its pH and osmolarity was adjusted to 7.4 with NaOH and 330 mOsm/L with sucrose, separately. Recording pipettes were pulled using a Sutter P-97 puller (Sutter Instruments, CA, USA), and its resistance was in the range of 3-6MΩ. The micropipette solution contained (in mM): 140 KCl, 2 MgCl 2 , 11 EGTA, 10 HEPES, 4 ATP, and 0.3 Na 2 GTP. Its pH and osmolarity was adjusted to 7.2 with KOH and 310 mOsm/L with sucrose, separately. After whole-cell configuration established, 70-80% series resistance and membrane capacitance current were compensated. The recording currents were sampled at 10 kHz and filtered at 2 kHz. DRG neurons (15-35μm in diameter) are used for electrophysiological recording. The membrane potential of neurons was clamped at −60 mV. Only DRG neuron with a resting membrane potential less than −50 mV was used for currentclamp recordings.

| Drug application
All drugs were obtained from Sigma (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 applied by gravity. The distance was ~30 μm between drug exit and recorded neurons. To block G protein and intracellular signal, some antagonists or blockers were dissolved in the internal solution and applied for intracellular dialysis through recording patch pipettes as described previously. 28,29 To ensure that dialysis drugs are infused into the cell interior, current recording was performed at least 30 minutes after cell membrane rupture.

| Data analysis
All data were expressed as mean ± SEM. The normality of the data distribution was analyzed by the Shapiro-Wilk test. If data were normally distributed, the data were statistically compared using Student's t test or analysis of variance (ANOVA), followed by Bonferroni's post hoc test. Nonlinear curve-fitting program ALLFIT was used for statistical analysis of concentration-response data.
In some DRG neurons, we pre-treated with DEX for 5min prior to the next I ATP recording. As shown in Figure 1B, DEX pretreatment (3 μM) decreased the peak amplitudes of both α,β-meATP-and ATP-activated currents. Figure 1C shows that the peak amplitudes of 100 μM α,β-meATP-activated currents decreased as the concentration of DEX increased from 0.1 μM to 10 μM in a representative DRG cell. Figure 1D shows concentration-effect curve of DEX on I ATP with an IC 50 (half-maximal effective concentration) value of 1.12 ± 0.16 μM. The results suggested that DEX inhibited P2X3 receptor-mediated ATP currents in a concentration-dependent manner.

| Concentration-response and current-voltage relationships for α,β-meATP in the absence and presence of DEX
We then studied whether the DEX-induced inhibition depended on the concentration of α,β-meATP. Concentration-response curves were plotted through a series of different concentration of α,β-meATP. Figure 2A shows that three representative ATP currents evoked by α,β-meATP at 3, 30, and 300 μM were decreased after DEX pretreatment (3 μM) for 5 min. Figure 2B shows that concentrationresponse curves for α,β-meATP were fit with the Hill equation in the absence and presence of DEX (3 μM). We observed that DEX pretreatment shifted downwards the concentration-response curve for α,β-meATP. First, maximal current response, which was evoked by 300 μM α,β-meATP, decreased 50.43 ± 4.75% with DEX (3 μM) pretreatment. Second, the Hill coefficient or slope of curves without and with DEX pretreatment was 1.40 ± 0.26 and 1.32 ± 0.37, respectively, with no significant difference (p > 0.1, Bonferroni's post hoc test). Third, DEX pretreatment did not change the EC 50 of α,β-meATP for P2X3 receptors, which were 29.45 ± 1.26 μM and 31.03 ± 1.49 μM, respectively, in the absence and presence of DEX (p > 0.1, Bonferroni's post hoc test). These results suggested that DEX could inhibit the maximum response to α,β-meATP, but not shift the sensitivity of P2X3 receptors to α,β-meATP.
To investigate whether the inhibition of ATP currents by DEX depended on membrane potentials, we observed the inhibitory effect of DEX on α,β-meATP-activated currents recorded at different clamping potentials. Figure 2C shows that DEX pretreatment (3 μM for 5min) inhibited the peak amplitudes of the three I ATP, which were evoked by 100 μM α,β-meATP when the membrane potential was clamped at −80 mV, −40 mV, and +20 mV, separately. Figure 2D shows the current-voltage (I-V) curves for α,β-meATP with and without DEX pretreatment. DEX did not change the reversal potential (near 0 mV) of the I-V curve, but decreased the slope of curve. There was no significant difference in the DEX-induced inhibition of ATP currents at different clamping potentials from −80 to 20 mV (p > 0.1, Bonferroni's post hoc test). The results indicated that DEX voltage independently inhibited P2X3 receptor-mediated ATP currents.

| DEX inhibits ATP currents via an α 2 -ARs, G i/o proteins and cAMP signaling pathway
As a selective α 2 -AR agonist, is the inhibitory effect of DEX on ATP currents mediated by α 2 -ARs? We examined the effects of the α 2 -AR antagonist yohimbine and the α 2A -AR antagonist BRL44408 on suppression of ATP currents by DEX. As shown in Figure 3A

| DEX suppresses α,β-meATP-evoked action potentials in rat DRG neurons
We further investigated the effect of DEX on action potentials  Figure 4D). The results suggested that DEX also suppressed α,β-meATP-evoked APs through α 2A -ARs in rat DRG neurons.

| DEX relieves α,β-meATP-induced nociceptive behaviors in rats
Finally, we investigated whether the suppression of P2X3 receptors by DEX in vitro played a role in the nociceptive behaviors induced by α,β-meATP in vivo. Figure 5A shows intraplantar injection of α,β-meATP (50 μg in 50 μl) caused an intense spontaneous flinch/shaking response in rats, which was attenuated by intraplantar pretreatment of DEX. DEX dose dependently (10, 30, and 100 ng) relieved the nociceptive behaviors induced by α,β-meATP (p < 0.05 and 0.01, one-way ANOVA followed by post hoc Bonferroni's test, n = 10; Figure 5A). The anti-nociceptive effect of 100 ng DEX was blocked by co-treated 150 ng BRL44408 (p < 0.01, one-way ANOVA followed by post hoc Bonferroni's test, n = 10; Figure 5A). In addition, 100 ng DEX had no effect on the α,β-meATP-induced nociceptive behaviors when injected into the contralateral paws. The results suggested that DEX had an anti-nociceptive effect on the α,β-meATPinduced nociceptive behaviors in vivo through peripheral α 2A -ARs.
The results suggested that DEX had also an analgesic effect on the α,β-meATP-induced mechanical allodynia through peripheral α 2A -ARs.

| DISCUSS ION
The present data demonstrated that the selective α 2 -AR agonist DEX The recorded ATP currents in the present experiments were mediated by P2X3 receptors, because they could be blocked by specifical antagonist of P2X3 and P2X2/3 receptor A-317491. 21 Moreover, α,β-meATP can only activate P2X3 and P2X1 receptors. 31 ATP receptors include P2X1-7 subtypes. Among all subtypes, P2X3 receptor subtype is mainly located in a subset of small-and medium-sized nociceptive DRG neurons. [17][18][19] The present study showed that DEX, a selective α 2A -AR agonist, concentration dependently inhibited P2X3 receptor-mediated ATP currents. The DEX-induced suppression did not alter the sensitivity of P2X3 receptor to α,β-meATP, but decreased the maximum response to α,β-meATP. DEX suppressed Intraplantar injection of P2X3 receptor agonists results in spontaneous pain behaviors and mechanical allodynia in rats. 25,45,46 Local pretreatment of α 1 -AR agonists augment P2X3 receptor-mediated flinching behaviors in rats. 47 This behavioral finding is consistent with F I G U R E 5 Relief of α,β-meATP-evoked nociceptive behaviors by DEX in rats. (A) Intraplantar injection of α,β-meATP (50μg in 50μl) caused spontaneous flinching behaviors in rats. Intraplantar pretreatment of DEX (10, 30, and 100 ng) dose dependently decreased the number of α,β-meATP-induced flinching behaviors. The anti-nociceptive effect of DEX (100ng) on the flinching behaviors was completely prevented by co-treatment of the α 2A -AR antagonist BRL44408 (BRL, 150 ng). DEX (100 ng) had no effect on α,β-meATP-induced flinching behaviors when it was injected into the contralateral hindpaw and α,β-meATP was injected into one hindpaw (100 ng contral). Bonferroni's post hoc test, *p < 0.05, **p < 0.01, compared with vehicle column; ## p < 0.01, compared with 100 ng DEX column. Each column represents the mean ±S.E.M. of 10 rats. (B) Intraplantar injection of α,β-meATP (50 μg in 50 μl) also caused a remarkable decrease in paw withdrawal thresholds (PWT, in g) at 0.5 and 2.5 h after injection and recovery at 24 h. The α,β-meATP-induced mechanical allodynia was significantly relieved by intraplantar pretreatment of DEX (100 ng), but not co-treatment of DEX (100 ng) and BRL44408 (BRL, 150 ng). *p < 0.05, **p < 0.01, Bonferroni's post hoc test, n = 10 rats in each group electrophysiological results that the activation of α1-ARs by noradrenaline potentiates ATP-evoked currents in DRG neurons by activating PKC. 26 The present results showed that peripheral pretreatment of the DEX dose dependently relieved the α,β-meATP-induced nociceptive behaviors. The anti-nociceptive effect of DEX occurred locally rather than systematically by directly activating peripheral α 2A -ARs

| CON CLUS IONS
Under inflammatory and neuropathic pain conditions, not only a large amount of ATP is released from damaged cells but also total expression and membrane expression of P2X3 receptors increases in DRG neurons. 25,[48][49][50] These P2X3 receptors, activated by released ATP, plays a prominent role in some pain states. 51 Our results suggested that DEX suppressed P2X3 receptor-mediated the electrophysiological activity and nociception through α 2A -ARs, revealing a peripheral novel mechanism underlying the analgesia of DEX.
Clinically, DEX, even when applied locally, may also effectively relieve pain involving peripheral P2X3 receptors.

CO N FLI C T O F I NTE R E S T
All authors declare no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
WPH designed this research. JWH, WLQ, QL, SW, TTL, and CYQ performed the experiments. JWH, WLQ, and QL participated in data analysis. JWH, WLQ, QL, and WPH wrote the paper. All authors contributed substantially to this research and reviewed this manuscript.

E TH I C A L A PPROVA L
The animal study was reviewed and approved by the animal research ethics committee of Hubei University of Science and Technology.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or used during the study appear in the submitted article.