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 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.
In the present experiments, α,β-meATP-induced ATP currents were recorded in small- and medium-sized DRG nociceptive neurons, where P2X3 homomeric and P2X2/3 heteromeric receptors are abundantly expressed [28, 27]. These recorded ATP currents were blocked by specific antagonist of P2X3 and P2X2/3 receptors, but not by antagonists of P2X4 receptors and P2X7 receptors, indicating they were P2X3 or P2X3-containing receptor-mediated currents. Moreover, α,β-meATP is only an activator of P2X3 and P2X1 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/A1 adenosine receptor signaling. Consistent with current results, other ion channels are 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 GABAA 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 . 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 was that the DRG neurons only express P2X3 receptors, but not A1 adenosine receptors.
The A1 adenosine receptor is coupled to Gi/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 the DRG neurons were intracellularly dialyzed with the Gi/o protein inhibitor PTX, the adenylate cyclase activator forskolin, or the cAMP analog 8Br-cAMP, indicating involvement of Gi/o-proteins and intracellular cAMP signaling. Our recent study has shown that CPA suppresses acid sensing ion channels via A1 adenosine receptors and intracellular Gi/o-proteins and cAMP signaling cascades in rat DRG neurons . 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 . Recent studies have reported that blockage of HCN channels inhibits the function of P2X2 and P2X3 receptors in rat DRG neurons via a cAMP-PKA signaling pathway . Leu-enkephalin inhibits P2X3 currents in DRG neurons through Gi/o-proteins, while it increases P2X3 currents via a PLC signaling pathway after pre-treatment of the neurons with a Gi/o-protein inhibitor PTX . The present and previous studies suggested P2X3 receptor was downstream target of PTX-sensitive Gi/o proteins and intracellular 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 . 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]. ATP activates P2X3 receptors and causes pain when injected into the skin . 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 receptors, 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 receptors 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 . 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.