RESUMO
P388D1 cells release arachidonic acid (AA) and produce prostaglandin E2 (PGE2) upon long-term stimulation with lipopolysaccharide (LPS). The cytosolic Group IVA (GIVA) phospholipase A2 (PLA2) has been implicated in this pathway. LPS stimulation also results in increased expression and secretion of a secretory PLA2, specifically GV PLA2. To test whether GV PLA2 contributes to PGE2 production and whether GIVA PLA2 activation increases the expression of GV PLA2, we utilized the specific GIVA PLA2 inhibitor pyrrophenone and second generation antisense oligonucleotides (AS-ONs) designed to specifically inhibit expression and activity of GV PLA2. Treatment of P388D1 cells with antisense caused a marked decrease in basal GV PLA2 mRNA and prevented the LPS-induced increase in GV PLA2 mRNA. LPS-stimulated cells release active GV PLA2 into the medium, which is inhibited to background levels by antisense treatment. However, LPS-induced PGE2 release by antisense-treated cells and by control cells are not significantly different. Collectively, the results suggest that the upregulation of GV PLA2 during long-term LPS stimulation is not required for PGE2 production by P388D1 cells. Experiments employing pyrrophenone suggested that GIVA PLA2 is the dominant player involved in AA release, but it appears not to be involved in the regulation of LPS-induced expression of GV PLA2 or cyclooxygenase-2.
Assuntos
Dinoprostona/fisiologia , Lipopolissacarídeos/metabolismo , Fosfolipases A/metabolismo , Regulação para Cima , Animais , Ácido Araquidônico/metabolismo , Linhagem Celular , Meios de Cultura/metabolismo , Ciclo-Oxigenase 2/metabolismo , DNA Complementar/metabolismo , Dinoprostona/metabolismo , Eicosanoides/metabolismo , Inibidores Enzimáticos/farmacologia , Fosfolipases A2 do Grupo V , Immunoblotting , Indóis/farmacologia , Lipídeos/química , Macrófagos/metabolismo , Camundongos , Oligonucleotídeos Antissenso/química , Fosfolipases A2 , Pirrolidinas/química , RNA/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , TransfecçãoRESUMO
Phospholipase A(2) (PLA(2)) appears to play a fundamental role in cell injury in the central nervous system. We have investigated PLA(2) expression in the astrocytoma cell line 1231N1, and found that GIVA, GIVB, GIVC and GVI PLA(2) messages are expressed. PLA(2) activity is increased by inflammatory/injury stimuli such as interleukin-1beta and lipopolysaccharide in these cells but with very different time courses. The arachidonic acid liberated is converted to prostaglandin E(2), possibly by cyclooxygenase-2, which is induced by inflammatory stimuli. This cell system emerges as a model to study injury/inflammation-related activation of the new PLA(2) forms GIVB and GIVC.