Ohsawa Keiko, Irino Yasuhiro, Nakamura Yasuko, Akazawa Chihiro, Inoue Kazuhide, Kohsaka Shinichi
Department of Neurochemistry, National Institute of Neuroscience, Kodaira, Tokyo 187-8502, Japan.
Glia. 2007 Apr 15;55(6):604-16. doi: 10.1002/glia.20489.
We previously reported that extracellular ATP induces membrane ruffling and chemotaxis of microglia and suggested that their induction is mediated by the Gi/o-protein coupled P2Y(12) receptor (P2Y(12)R). Here we report discovering that the P2X(4) receptor (P2X(4)R) is also involved in ATP-induced microglial chemotaxis. To understand the intracellular signaling pathway downstream of P2Y(12)R that underlies microglial chemotaxis, we examined the effect of two phosphatidylinositol 3'-kinase (PI3K) inhibitors, wortmannin, and LY294002, on chemotaxis in a Dunn chemotaxis chamber. The PI3K inhibitors significantly suppressed chemotaxis without affecting ATP-induced membrane ruffling. ATP stimulation increased Akt phosphorylation in the microglia, and the increase was reduced by the PI3K inhibitors and a P2Y(12)R antagonist. These results indicate that P2Y(12)R-mediated activation of the PI3K pathway is required for microglial chemotaxis in response to ATP. We also found that the Akt phosphorylation was reduced when extracellular calcium was chelated, suggesting that ionotropic P2X receptors are involved in microglial chemotaxis by affecting the PI3K pathway. We therefore tested the effect of various P2X(4)R antagonists on the chemotaxis, and the results showed that pharmacological blockade of P2X(4)R significantly inhibited it. Knockdown of the P2X(4) receptor in microglia by RNA interference through the lentivirus vector system also suppressed the microglial chemotaxis. These results indicate that P2X(4)R as well as P2Y(12)R is involved in ATP-induced microglial chemotaxis.
我们之前报道过，细胞外ATP可诱导小胶质细胞的膜皱襞形成和趋化作用，并表明其诱导作用是由Gi/o蛋白偶联的P2Y(12)受体（P2Y(12)R）介导的。在此我们报告发现P2X(4)受体（P2X(4)R）也参与ATP诱导的小胶质细胞趋化作用。为了解P2Y(12)R下游介导小胶质细胞趋化作用的细胞内信号通路，我们在Dunn趋化小室中检测了两种磷脂酰肌醇3'-激酶（PI3K）抑制剂渥曼青霉素和LY294002对趋化作用的影响。PI3K抑制剂显著抑制趋化作用，而不影响ATP诱导的膜皱襞形成。ATP刺激可增加小胶质细胞中Akt的磷酸化，PI3K抑制剂和P2Y(12)R拮抗剂可减少这种增加。这些结果表明，P2Y(12)R介导的PI3K通路激活是小胶质细胞对ATP作出趋化反应所必需的。我们还发现，当细胞外钙被螯合时，Akt磷酸化减少，这表明离子型P2X受体通过影响PI3K通路参与小胶质细胞趋化作用。因此，我们检测了各种P2X(4)R拮抗剂对趋化作用的影响，结果表明P2X(4)R的药理学阻断显著抑制了趋化作用。通过慢病毒载体系统进行RNA干扰敲减小胶质细胞中的P2X(4)受体也抑制了小胶质细胞趋化作用。这些结果表明，P2X(4)R以及P2Y(12)R都参与ATP诱导的小胶质细胞趋化作用。
