P2Y13 Receptor-Mediated Rapid Increase in Intracellular Calcium Induced by ADP in Cultured Dorsal Spinal Cord Microglia

Abstract

P2Y receptors have been implicated in the calcium mobilization by the response to neuroexcitatory substances in neurons and astrocytes, but little is known about P2Y receptors in microglia cells. In the present study, the effects of ADP on the intracellular calcium concentration ([Ca(2+)]i) in cultured dorsal spinal cord microglia were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescence indicator that could monitor real-time alterations of [Ca(2+)]i. Here we show that ADP (0.01-100 μM) causes a rapid increase in [Ca(2+)]i with a dose-dependent manner in cultured microglia. The action of ADP on [Ca(2+)]i was significantly blocked by MRS2211 (a selective P2Y13 receptor antagonist), but was unaffected by MRS2179 (a selective P2Y1 receptor antagonist) or MRS2395 (a selective P2Y12 receptor antagonist), which suggest that P2Y13 receptor may be responsible for ADP-evoked Ca(2+) mobilization in cultured microglia. P2Y13-evoked Ca(2+) response can be obviously inhibited by BAPTA-AM and U-73122, respectively. Moreover, removal of extracellular Ca(2+) (by EGTA) also can obvious suppress the Ca(2+) mobilization. These results means both intracellular calcium and extracellular calcium are potentially important mechanisms in P2Y13 receptor-evoked Ca(2+) mobilization. However, P2Y13 receptor-evoked Ca(2+) response was not impaired after CdCl2 and verapamil administration, which suggest that voltage-operated Ca(2+) channels may be not related with P2Y13-evoked Ca(2+) response. In addition, Ca(2+) mobilization induced by ADP was abolished by different store-operated Ca(2+) channels (SOCs) blocker, 2-APB (50 μM) and SKF-96365 (1 mM), respectively. These observations suggest that the activation of P2Y13 receptor might be involved in the effect of ADP on [Ca(2+)]i in cultured dorsal spinal cord microglia. Furthermore, our results raise a possibility that P2Y13 receptor activation causes Ca(2+) release from Ca(2+) store, which leads to the opening of SOCs.

P2Y₁₂and P2Y₁₃ receptors involved in ADPβs induced the release of IL-1β, IL-6 and TNF-α from cultured dorsal horn microglia

“…We found that some previous studies support the notion that P2Y 1 and P2Y 11 receptors are not present in dorsal spinal cord microglia cells. 27 , 38 , 39 Furthermore, Horváth et al discerned that in inflammatory pain rat models, P2Y 12 receptor antagonist did not change the increased level of IL-10 after CFA stimulus. 35 From these observations, we conclude that P2Y 12 and P2Y 13 receptor activation does not lead to increased level of IL-10 in cultured dorsal spinal cord microglia cells.…”

“…According to some previous studies, 25 – 27 microglia cells were pretreated with P2Y 12 receptor antagonist MRS2395 (10 μM) and/or P2Y 13 receptor antagonist MRS2211 (10 μM) for 20 min to test the effect of receptor inhibition on ADPβs-induced downstream effects. In some previous studies, Y-27632, SB203580 and PDTC at 5–20 μM were frequently used in order to establish the involvement of ROCK, P38MAPK and NF-κB signaling in cellular responses.…”

A Selective P2Y Purinergic Receptor Agonist 2-MesADP Enhances Locomotor Recovery after Acute Spinal Cord Injury

“…Many responses of purinergic receptors are related to synaptic signal transmission, the interaction between neuron/glia and vascular cells, neurodegenera- tion, inflammatory cells, and neuronal apoptosis. P2Y receptors coupled with a secondary messenger signaling cascade, the GPCRs, which stimulates several intercellular signal pathways, resulting in cell proliferation [23], maturation, and neurotransmission [24]. On the other hand, activation of P2Y receptors could induce Ca 2+ wave and then activate downstream signaling pathways, such as protein kinase C, extracellular signal-regulated kinase, mitogen-activated protein kinases, c-Fos/c-Jun signaling [25], PI3-K/Akt/ Creb pathway, and Jak2/Stat3, Sapk/Jnk signaling [26].…”

P2Y13 receptors regulate microglial morphology, surveillance, and resting levels of interleukin 1β release

“…We suggest above that some of the effects of P2Y 13 deletion may reflect an increase of cAMP concentration in the cell, but other signaling pathways may also be associated with the P2Y 13 receptor. These include G q ‐coupled elevations of intracellular [Ca 2+ ] mediated by internal store release and activation of store‐operated calcium channels (Carrasquero et al, ; Zeng et al, ), Erk1/2 activation (Ortega, Perez‐Sen, Delicado, & Teresa Miras‐Portugal, ), and regulation of transcription through a rise in nuclear calcium level and activation of nuclear factor erythroid‐derived 2‐like 2, a transcription factor that regulates the oxidative stress response (Espada et al, ; Lyubchenko et al, ). P2Y 13 may also inhibit Akt (Chatterjee & Sparks, ), which is an important mediator of P2Y 12 ‐mediated actin re‐organization.…”

“…P2Y 13 mRNA is upregulated in pathological conditions such as demyelination (Bedard, Tremblay, Chernomoretz, & Vallieres, 2007) and neuropathic pain (Kobayashi, Yamanaka, Yanamoto, Okubo, & Noguchi, 2012;Niu et al, 2017), suggesting a role of this receptor in neuroinflammation. However, its function in microglia, especially under physiological conditions, is still unclear, although it may evoke a rise of [Ca 2+ ] i in response to ADP (Zeng et al, 2014) and be involved in the release of proinflammatory cytokines (Liu et al, 2017). Increasing evidence also supports the notion that ADP may act through P2Y 13 receptors to regulate the structural complexity of microglia (Matyash, Zabiegalov, Wendt, Matyash, & Kettenmann, 2017;Stefani et al, 2018) and contribute to the homeostatic control of adult hippocampal neurogenesis in situ (Stefani et al, 2018).…”