CB1 Receptors Mediated Inhibition of ATP-Induced [Ca2+]i Increase in Cultured Rat Spinal Dorsal Horn Neurons

Abstract

Spinal cannabinoid receptor 1 (CBR) and purinergic P2X receptors (P2XR) play a critical role in the process of pathological pain. Both CBR and P2XR are expressed in spinal dorsal horn (DH) neurons. It is not clear whether CB receptor activation modulates the function of P2X receptor channels within dorsal horn. For this reason, we observed the effect of CP55940 (cannabinoid receptor agonist) on ATP-induced Ca mobilization in cultured rat DH neurons. The changes of intracellular calcium concentration ([Ca]i) were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator. 100 μM ATP caused [Ca]i increase in cultured DH neurons. ATP-evoked [Ca]i increase in DH neurons was blocked by chelating extracellular Ca and P2 purinoceptor antagonist PPADS. At the same time, ATP-γ-S (a non-hydrolyzable ATP analogue) mimicked the ATP action, while P2Y receptor agonist ADP failed to evoke [Ca]i increase in cultured DH neurons. These data suggest that ATP-induced [Ca]i elevation in cultured DH neurons is mediated by P2X receptor. Subsequently, we noticed that, in cultured rat DH neurons, ATP-induced Ca mobilization was inhibited after pretreated with CP55940 with a concentration-dependent manner, which implies that the opening of P2X receptor channels are down-regulated by activation of cannabinoid receptor. The inhibitory effect of CP55940 on ATP-induced Ca response was mimicked by ACEA (CBR agonist), but was not influenced by AM1241 (CBR agonist). Moreover, the inhibitory effect of CP55940 on ATP-induced Ca mobilization was blocked by AM251 (CB receptor antagonist), but was not influenced by AM630 (CB receptor antagonist). In addition, we also observed that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a cell-permeable cAMP analog) reversed the inhibitory effect of CP55940, respectively. In a summary, our observations raise a possibility that CBR rather than CBR can downregulate the opening of P2X receptor channels in DH neurons. The reduction of cAMP/PKA signaling is a key element in the inhibitory effect of CBR on P2X-channel-induced Ca mobilization.

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

“…Similarly, cannabinoid inhibits ATP‐induced currents and calcium influx through G i/o protein‐coupled CB1 cannabinoid receptors and AC‐cAMP‐PKA signaling pathway in rat trigeminal ganglionic neurons and cultured spinal dorsal horn neurons. 42 , 43 In addition, intracellular application of BAPTA, a chelator of calcium ions, failed to reverse the inhibitory effect of DEX on α,β‐meATP‐induced currents, suggesting no involvement of intracellular Ca. 2 +…”

CB2/miR-124 signaling down-regulate the expression of purinergic P2X4 and P2X7 receptor in dorsal spinal cord of CCI rats

“…The analgesic effect of CB1 receptor activation is mediated by a negative modulation of the P2X 3 receptor in the primary afferent neurons in carrageenan-induced inflammatory pain in mice [12]. In cultured dorsal spinal neurons, the opening of P2X receptor channels is down-regulated by activation of cannabinoid CB1 receptor [13]. Our previous studies indicated that CB2 receptor activation produces a pronounced inhibition of CCI-induced thermal hyperalgesia and significantly inhibited the increased expression of P2Y 12 and P2Y 13 receptors, which open up the possibility that P2Y 12 and P2Y 13 receptor expression are down-regulated by CB2 receptor activation [11].…”

“…In cultured dorsal spinal neurons, the opening of P2X receptor channels is down-regulated by activation of cannabinoid CB1 receptor [13]. Our previous studies indicated that CB2 receptor activation produces a pronounced inhibition of CCI-induced thermal hyperalgesia and significantly inhibited the increased expression of P2Y 12 and P2Y 13 receptors, which open up the possibility that P2Y 12 and P2Y 13 receptor expression are down-regulated by CB2 receptor activation [11]. Based on these reports, we hypothesized that the expression of P2X 4 and P2X 7 receptor in dorsal spinal cord may be also influenced after CB2 receptor activation in neuropathic pain rats.…”