SB-203347

Arachidonic acid (AA) mobilization by phospholipase A2 (PLA2) and subsequent prostaglandin synthesis is considered to be a pivotal event in inflammation. The purpose of this study was to assess the efficacy of a Type II PLA2 specific inhibitor, SB 203347, in reducing prostaglandin production in Type II PLA2-transfected Chinese hamster ovary (CHO) cells and in human placenta. In both experimental models utilised, Type II PLA2 represents the principal isozyme contributing to total PLA2 enzymatic activity. PLA2 enzymatic activity released into cell culture media and placental explant media was quantified by radiolabelled substrate assay [14C-phosphatidylethanolamine (PE)]. Immunoreactive prostaglandin F2alpha (PGF2alpha) concentrations were determined by radioimmunoassay. SB 203347 (at 0.1-10 microM final concentration) inhibited PLA2 enzymatic activity released by Zn++ -activated CHO cells by up to 60% (P<0.0001). The concentration of PGF2alpha present in culture media was concomitantly reduced by up to 90% (P<0.0001). Similar results were observed for human placental explants. Treatment of human placental explants with SB 203347 (1 microM final concentration) significantly reduced PLA2 enzymatic activity recovered in media after 24 h incubation (P<0.0001; n = 10). Incubation media PGF2alpha concentrations were also reduced by 60% (P<0.00001). The addition of endogenous arachidonic acid (30 microM final concentration) significantly attenuated SB 203347-inhibition of PGF2alpha release (P<0.01). The data obtained in this study are consistent with the hypothesis that Type II PLA2 contributes to the liberation of arachidonic acid for prostanoid formation in human placenta and in cells that abundantly express this isozyme.

Human monocytes possess both the cytosolic 85 kDa phospholipase (PLA) A2 and a 14 kDa PLA2 and are capable of simultaneously producing prostanoids (PG), leukotrienes (LT) and platelet activating factor (PAF). As the exact roles of the two enzymes in monocyte lipid mediator formation was unclear, both selective PLA2 inhibitors and antisense were used to elucidate their respective roles. Reduction in 85 kDa PLA2 cellular protein levels by initiation site-directed antisense (SK 7111) or exposure to the 85 kDa PLA2 inhibitor, arachidonyl trifluormethyl ketone (AACOCF3), prevented A23187 or zymosan-stimulated monocytes prostanoid formation but not LTC4 or PAF production. This confirmed the important role of the 85 kDa PLA2 in prostanoid formation but indicated a less significant role in LT or PAF biosynthesis. Alternatively, treatment of monocytes with the selective, active-site-directed 14 kDa PLA2 inhibitor, SB 203347, totally inhibited LT and PAF formation, while prostanoid formation was not altered. Addition of 20 uM exogenous arachidonic acid (AA) to monocytes exposed to SB 203347 did not alter A23187-induced LTC4 generation, indicating that SB 203347 had no effect on downstream AA metabolizing enzymes in this setting. Taken together, these results provide evidence that the 14 kDa PLA2 provides substrate for monocyte LT and PAF formation, while the 85 kDa PLA2 plays a more significant role in the formation of PG.