INVOLVEMENT OF PLATELET-ACTIVATING-FACTOR IN CELL-DEATH INDUCED UNDERISCHEMIA POSTISCHEMIA-LIKE CONDITIONS IN AN IMMORTALIZED HIPPOCAMPAL CELL-LINE/

The involvement of platelet-activating factor (PAF) in cell damage ind uced by ischemia/postischemia-like conditions was studied in a hippoca mpus-derived cell line, HN33.11. Cells exposed to N-2-saturated glucos e-free HEPES-buffered saline (ischemia) for 5 h followed by 18 h of in cubation in serum-free control medium (postischemia reincubation) rema ined 67.4 +/- 2.4% viable in comparison with sham-treated cells. Analy sis of DNA fragmentation in combination with Hoechst 33258 staining in dicates that apoptosis is the dominant mode of cell death in the prese nt model. PAF level during 10 h of ischemia was unchanged. However, an increase in PAF accumulation was found early during the reincubation period that followed 5 h of ischemia. Peak PAF concentrations were not ed at 2 h after initiation of reincubation and rapidly declined to con trol level after 7 h of reincubation. Consistent with a role of PAF in mediating cell death under ischemia/postischemia reincubation in this model, the PAF antagonist BN 50739 exerted a dose-dependent protectiv e effect, Maximal protection (85.7 +/- 5.4%) of the cells from ischemi a/reincubation-induced cell damage was achieved at 0.1 mu M BN 50739. The PAF antagonist lacked any protective effect against ischemia-induc ed cell death, On the other hand, the addition of the stable PAF analo gue cyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (MC-PAF) at the onset of ischemia potentiated ischemia/reincubation-induced apoptosis -an effect that was blocked by BN 50739. Pretreatment of HN33.11 cells with the Ca2+ chelator 1,2-bis (2-aminophenoxy) ethane-N,N, N,N-tetra acetic acid acetoxymethyl ester (BAPTA-AM) also provided a protective effect against ischemia/reincubation-induced cell damage. BAPTA-AM inc reased cell viability by 50%. Pretreatment with BAPTA-AM also decrease d ischemia/reincubation-induced PAF accumulation in HN33.11 cells. The results suggest that PAF, acting via a PAF receptor, is at least in p art mediating apoptosis under ischemia/postischemia-like conditions in HN33.11 cells.