Involvement of P2X4 and P2Y12 receptors in ATP-induced microglial chemotaxis.
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.
Ohsawa
Keiko
K
Department of Neurochemistry, National Institute of Neuroscience, Kodaira, Tokyo 187-8502, Japan.
Irino
Yasuhiro
Y
Nakamura
Yasuko
Y
Akazawa
Chihiro
C
Inoue
Kazuhide
K
Kohsaka
Shinichi
S
D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D002633:Chemotaxis / Q000187:drug effects / Q000502:physiology*; D015536:Down-Regulation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008565:Membrane Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D051058:Oncogene Protein v-akt / Q000187:drug effects / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D034622:RNA Interference / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058848:Receptors, Purinergic P2X4; D058925:Receptors, Purinergic P2Y12; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
D000255:Adenosine Triphosphate; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D051058:Oncogene Protein v-akt; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058848:Receptors, Purinergic P2X4; D058925:Receptors, Purinergic P2Y12