Antisense oligonucleotide inhibition of type II phospholipase A(2) expression, release and activity in vitro

Phospholipase A(2) (PLA(2)) enzymes that regulate the release of arachidoni c acid from cell membrane phospholipids represent a crucial rate-limiting s tep for the prostaglandin biosynthetic pathway. The aim of this study was t o determine the mechanism of action and effects of type II PLA(2) antisense oligonucleotides on type II PLA, mRNA relative abundance, and the release of PLA(2) enzymatic activity and prostaglandin F-2 alpha (PGF(2 alpha)) in vitro. A human placental explant system was used to evaluate the effects of the type II PLA(2) specific antisense oligonucleotides A (5 ' -GGGTGGGTATA GAAGGGCTCC-3 ', complementary to the base sequence 697-717 of the type II P LA(2) gene) and B (5 ' -TTTTTGATTTGCTAATTGCTT-3 ', complementary to the bas e sequence 821-841 of the type II PLA(2) gene). PLA(2) activity released fr om explants was quantified by radiolabelled substrate assay using C-14-phos phatidylcthanolamine (PE), and PGF(2 alpha) content was analyzed by radioim munoassay. Compared with control, the release of PLA(2) activity and PGF(2 alpha) was significantly reduced over the 24-h period by treatment with bot h antisense oligonucleotides (P < 0.05). At this concentration, type II PLA (2) mRNA abundance was also significantly reduced by both antisense oligonu cleotides A and B (P <0.05). This data demonstrates the efficacy of antisen se oligonucleotide inhibition of secretory PLA(2) (sPLA(2)) expression and activity, and the contribution of sPLA(2) to placental prostilglandin produ ction. (C) 2001 Harcourt Publishers Ltd.