ATP-stimulated Ca2+ influx and phospholipase D activities of a rat brain-derived type-2 astrocyte cell line, RBA-2, are mediated through P2X7 receptors.
This study characterizes and examines the P2 receptor-mediated signal transduction pathway of a rat brain-derived type 2 astrocyte cell line, RBA-2. ATP induced Ca2+ influx and activated phospholipase D (PLD). The ATP-stimulated Ca2+ influx was inhibited by pretreating cells with P2 receptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), in a concentration-dependent manner. The agonist 2'- and 3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) stimulated the largest increases in intracellular Ca2+ concentrations ([Ca2+]i); ATP, 2-methylthioadenosine triphosphate tetrasodium, and ATPgammaS were much less effective, whereas UTP, ADP, alpha,beta-methylene-ATP, and beta,gamma-methylene-ATP were ineffective. Furthermore, removal of extracellular Mg2+ enhanced the ATP- and BzATP-stimulated increases in [Ca2+]i. BzATP stimulated PLD in a concentration- and time-dependent manner that could be abolished by removal of extracellular Ca2+ and was inhibited by suramin, PPADS, and oxidized ATP. In addition, PLD activities were activated by the Ca2+ mobilization agent, ionomycin, in an extracellular Ca2+ concentration-dependent manner. Both staurosporine and prolonged phorbol ester treatment inhibited BzATP-stimulated PLD activity. Taken together, these data indicate that activation of the P2X7 receptors induces Ca2+ influx and stimulates a Ca2+-dependent PLD in RBA-2 astrocytes. Furthermore, protein kinase C regulates this PLD.
Sun
S H
SH
Institute of Neuroscience, National Yang Ming University, Taipei, Taiwan, ROC.
Lin
L B
LB
Hung
A C
AC
Kuo
J S
JS
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology*; D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D001921:Brain / Q000166:cytology*; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D008274:Magnesium / Q000008:administration & dosage; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D058919:Purinergic P2 Receptor Antagonists; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D058486:Receptors, Purinergic P2X7
C546470:P2rx7 protein, rat; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D011732:Pyridoxal Phosphate; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D010739:Phospholipase D; D008274:Magnesium; D002118:Calcium