Abstract
The aim of this study was to characterize the regulatory mechanisms of the P2X 7 receptor (P2X 7R)-mediated phospholipase D (PLD) activation in a rat brain-derived Type-2 astrocyte cell line, RBA-2. A time course study revealed that activation of P2X 7R resulted in a choline and not phosphorylcholine formation, suggesting that activation of P2X 7R is associated with the phosphatidylcholine–PLD (PC–PLD) in these cells. GF 109203X, a selective protein kinase C (PKC) inhibitor, partially inhibited the P2X 7R-mediated PLD activation, while blocking the phorbol 12-myristate 13-acetate (PMA)-stimulated PLD activity. In addition, PMA synergistically activated the P2X 7R-mediated PLD activity. Furthermore, genistein, a tyrosine kinase inhibitor, blocked the P2X 7R-activated PLD, while KN62, a Ca 2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor, was less effective, whereas the mitogen-activated protein kinase (MAPK) inhibitor PD98059 was ineffective. No additive inhibitory effects were found by simultaneous treatment of GF 109203X and KN62 on P2X 7R-activated PLD. Taken together, these results demonstrate that both PKC-dependent and PKC-independent signaling pathways are involved in the regulation of P2X 7R-mediated PLD activation. Additionally, CaMKII may participate in the PKC-dependent pathway, and tyrosine kinase may play a pivotal role on both PKC-dependent and PKC-independent pathways in the P2X 7R-mediated PLD activation in RBA-2 cells.
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