P2Y2 nucleotide receptors inhibit trauma-induced death of astrocytic cells.
Nucleotides as well as other neurotransmitters are known to be released to the extracellular space upon injury. To determine whether nucleotides acting on P2Y(2) nucleotide receptors promote protective or degenerative events after trauma in astrocytic cells, a well-established model of in vitro brain trauma was applied to 1321N1 cells expressing recombinant P2Y(2) nucleotide receptors (P2Y(2)R-1321N1). Cellular death was examined by measuring DNA fragmentation and caspase activation. Fragmented DNA was observed 48 h post-injury in 1321N1 cells, while P2Y(2) nucleotide receptor expressing cells did not show DNA fragmentation. A laddering pattern of fragmented DNA following injury was observed upon inhibition of P2Y(2) nucleotide receptors with suramin. Time-dependent increases of cleaved caspase-9, a mitochondrial-associated caspase, correlated with injury-induced cellular death. A decreased bax/bcl-2 gene expression ratio was observed in P2Y(2)R-1321N1 cells after traumatic injury, while untransfected 1321N1 cells showed a significant time-dependent increase of the bax/bcl-2 gene expression ratio. Activation of protein kinases was assessed to determine the signaling pathways involved in cell death and survival responses following traumatic injury. In P2Y(2)R-1321N1 and 1321N1 cells p38 phosphorylation was stimulated in a time-dependent manner but the phosphatidylinositol 3-kinase-dependent activation of extracellular signal-regulated kinase 1/2 and protein kinase B (PKB)/Akt was only observed in P2Y(2)R-1321N1 cells after injury. The stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) signaling pathway was not activated by traumatic injury in either astrocytic cell line. Inhibition of p38 kinase signaling pathway by treatment with PD1693, a MKK3/6 inhibitor, abolished the expression of cleaved caspase-9, the increase in the bax/bcl-2 gene expression ratio, as well as the fragmentation of DNA that followed injury of 1321N1 cells. Taken together, our results demonstrate a novel role for P2Y(2) nucleotide receptors and extracellular nucleotides in mediating survival responses to glial cells undergoing cellular death induced by trauma.
Burgos
Michelle
M
Department of Biochemistry, Medical-Sciences Campus, University of Puerto Rico, San Juan, Puerto Rico.
Neary
Joseph T
JT
González
Fernando A
FA
D000704:Analysis of Variance; D001253:Astrocytes / Q000187:drug effects / Q000502:physiology*; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002461:Cell Line, Transformed; D053938:DNA Fragmentation; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D058473:Receptors, Purinergic P2Y2; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013498:Suramin / Q000633:toxicity; D014162:Transfection / Q000379:methods; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism
D004791:Enzyme Inhibitors; D007093:Imidazoles; C546385:P2RY2 protein, human; D019253:Proto-Oncogene Proteins c-bcl-2; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; D051028:bcl-2-Associated X Protein; D013498:Suramin; D011494:Protein Kinases; C408604:2-(4-nitrophenyl)-4-(4-fluorophenyl)-5-(4-pyridinyl)-1H-imidazole