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.

Lysophosphatidic acid (LPA), a lipid metabolite, plays a role in both neuropathic and inflammatory pain through LPA1 receptors. P2X3 receptor has also been shown to participate in these pathological processes. However, it is still unclear whether there is a link between LPA signaling and P2X3 receptors in pain. Herein, we show that a functional interaction between them in rat dorsal root ganglia (DRG) neurons. Pretreatment of LPA concentration-dependently enhanced α,β-methylene-ATP (α,β-meATP)-induced inward currents mediated by P2X3 receptors. LPA significantly increased the maximal current response of α,β-meATP, showing an upward shift of the concentration-response curve for α,β-meATP. The LPA enhancement was independent on the clamping-voltage. Enhancement of P2X3 receptor-mediated currents by LPA was prevented by the LPA1 receptor antagonist Ki16198, but not by the LPA2 receptor antagonist H2L5185303. The LPA-induced potentiation was also attenuated by intracellular dialysis of either G-protein inhibitor or protein kinase C (PKC) inhibitor, but not by Rho inhibitor. Moreover, LPA significantly changed the membrane potential depolarization and action potential burst induced by α,β-meATP in DRG neurons. Finally, LPA exacerbated α,β-meATP- induced nociceptive behaviors in rats. These results suggested that LPA potentiated the functional activity of P2X3 receptors in rat primary sensory neurons through activation of the LPA1 receptor and its downstream PKC rather than Rho signaling pathway, indicating a novel peripheral mechanism underlying the sensitization of pain.

Purinergic signaling is involved in multiple pain processes. P2X3 receptor is a key target in pain therapeutics, while A1 adenosine receptor signaling plays a role in analgesia. However, it remains unclear whether there is a link between them in pain. The present results showed that the A1 adenosine receptor agonist N 6 -cyclopentyladenosine (CPA) concentration-dependently suppressed P2X3 receptor-mediated and α,β-methylene-ATP (α,β-meATP)-evoked inward currents in rat dorsal root ganglion (DRG) neurons.CPA signi cantly decreased the maximal current response of α,β-meATP, as shown a downward shift of its concentration-response curve. The CPA-induced suppression was independent on the clampingvoltageof the membrane. Inhibition of ATP currents by CPA was completely prevented by the A1 adenosine receptor antagonist KW-3902, and disappeared after the intracellular dialysis of either the G i/oprotein inhibitor pertussis toxin, the adenylate cyclase activator forskolin, or the cAMP analog 8-Br-cAMP.Morover, CPA suppressed the membrane potential depolarization and action potential burst induced by α,β-meATP in DRG neurons. Finally, CPA relieved α,β-meATP-induced nociceptive behaviors in rats by activating peripheral A1 adenosine receptors in dose-dependent manner. These results indicated that CPA inhibited P2X3 receptor activity in rat primary sensory neurons by activating A1 adenosine receptors, G i/oproteins and intracellular cAMP signaling, revealing a novel peripheral mechanism underlying its analgesic effect.

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Lysophosphatidic acid (LPA) is a phospholipid which has been implicated in pain. Acid‐sensing ion channels (ASICs) are important players in pain associated with tissue acidification. However, it is still unclear whether there is a link between LPA signaling and ASICs in pain processes. Herein, we show that a functional interaction between them in rat dorsal root ganglia (DRG) neurons. Pre‐application of LPA enhanced ASIC‐mediated and acid‐evoked inward currents in a concentration‐dependent manner. LPA shifted the concentration–response curve for protons upwards, with an increase of 41.79 ± 4.71% in the maximal current response of ASICs to protons in the presence of LPA. Potentiation of ASIC currents by LPA was blocked by the LPA1 receptor antagonist Ki16198, but not by the LPA2 receptor antagonist H2L5185303. The LPA‐induced potentiation was also prevented by intracellular application of either G protein inhibitor or protein kinase C (PKC) inhibitor, but not by Rho inhibitor. LPA also enhanced ASIC3 currents in CHO cells co‐expressing ASIC3 and LPA1 receptors, but not in cells expressing ASIC3 alone. Moreover, LPA increased the amplitude of the depolarization and the number of spikes induced by acid stimuli. Finally, LPA exacerbated acid‐induced nociceptive behaviors in rats. These results suggested that LPA enhanced ASIC‐mediated electrophysiological activity and nociception via a LPA1 receptor and its downstream PKC rather than Rho signaling pathway, which provided a novel peripheral mechanism underlying the sensitization of pain.

Purinergic signaling is involved in multiple pain processes. P2X3 receptor is a key target in pain therapeutics, while A1 adenosine receptor signaling plays a role in analgesia. However, it remains unclear whether there is a link between them in pain. The present results showed that the A1 adenosine receptor agonist N6-cyclopentyladenosine (CPA) concentration-dependently suppressed P2X3 receptor-mediated and α,β-methylene-ATP (α,β-meATP)-evoked inward currents in rat dorsal root ganglion (DRG) neurons. CPA significantly decreased the maximal current response of α,β-meATP, as shown a downward shift of its concentration-response curve. The CPA-induced suppression was independent on the clamping-voltageof the membrane. Inhibition of ATP currents by CPA was completely prevented by the A1 adenosine receptor antagonist KW-3902, and disappeared after the intracellular dialysis of either the Gi/o-protein inhibitor pertussis toxin, the adenylate cyclase activator forskolin, or the cAMP analog 8-Br-cAMP. Morover, CPA suppressed the membrane potential depolarization and action potential burst induced by α,β-meATP in DRG neurons. Finally, CPA relieved α,β-meATP-induced nociceptive behaviors in rats by activating peripheral A1 adenosine receptors in dose-dependent manner. These results indicated that CPA inhibited P2X3 receptor activity in rat primary sensory neurons by activating A1 adenosine receptors, Gi/o-proteins and intracellular cAMP signaling, revealing a novel peripheral mechanism underlying its analgesic effect.

Lysophosphatidic acid (LPA), a lipid metabolite, plays a role in both neuropathic and inflammatory pain through LPA1 receptors. P2X3 receptor has also been shown to participate in these pathological processes. However, it is still unclear whether there is a link between LPA signaling and P2X3 receptors in pain. Herein, we show that a functional interaction between them in rat dorsal root ganglia (DRG) neurons. Pre-application of LPA enhanced P2X3 receptor-mediated and α,β-methylene-ATP (α,β-meATP)-evoked inward currents in a concentration-dependent and voltage-independent manner. LPA shifted the concentration-response curve for α,β-meATP upwards, with an increase of 44.18 ± 8.74% in the maximal current response of P2X3 receptors to α,β-meATP in the presence of LPA. Enhancement of α,β-meATP-evoked currents by LPA was blocked by the LPA1 receptor antagonist Ki16198, but not by the LPA2 receptor antagonist H2L5185303. The LPA-induced potentiation was also prevented by intracellular application of either G- protein inhibitor or protein kinase C (PKC) inhibitor, but not by Rho inhibitor. Moreover, LPA increased the membrane excitability of DRG neurons, with a significant increase in the amplitude of the depolarization and the number of spikes induced by α,β-meATP in the presence of LPA. Finally, LPA exacerbated α,β-meATP- induced nociceptive behaviors in rats. These results suggested that LPA enhanced the functional activity of P2X3 receptors in rat primary sensory neurons through a mechanism that depends on the activation of the LPA1 receptor and its downstream PKC rather than Rho signaling pathway, indicating a novel peripheral mechanism underlying the sensitization of pain.

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