Platelet‐activating factor (PAF) may be a neuromodulator involved in neural cell differentiation, cerebral inflammation, and ischemia. The PAF receptor is a member of the G protein‐coupled receptor superfamily. In the present study, we sought to define the specific G protein(s) that mediate PAF‐stimulated phosphoinositide (PI) metabolism in an immortalized hippocampal cell line, HN33.11. PAF increased the production of 3H‐labeled inositol phosphates (IPs) with EC50 values of 1.2–1.5 nM. The effect of PAF on 3H‐IPs formation was completely blocked by the PAF antagonist BN 50739 at a concentration of 300 nM. Pertussis toxin pretreatment attenuated PAF‐stimulated 3H‐IPs production by 20–30% (p < 0.05). Consistent with a role for Gi1/2 in this response, antiserum against Gαi1/2 blocked the response to a similar degree. Pretreatment of permeabilized cells with Gαq/11 antiserum attenuated the response by 70% (p < 0.05), suggesting a role for Gq/11 in mediating the PAF response in this cell line. Stimulation with PAF increased [α‐32P]‐GTP binding to both Gαq and Gαi1/2 proteins. Moreover, specific [3H]PAF binding sites coprecipitated with Gαq and Gαi1/2 proteins. The results suggest that PAF‐stimulated PI metabolism in HN33.11 cells is mediated by both Gq and Gi1/2 proteins.

Abstract:The involvement of platelet-activating factor (PAF) in cell damage induced by ischemia/postischemialike conditions was studied in a hippocampus-derived cell line, HN33.11. Cells exposed to N 2-saturated glucose-free HEPES-buffered saline (ischemia) for 5 h followed by 18 h of incubation in serum-free control medium (postischemia reincubation) remained 67.4 ±2.4% viable in comparison with sham-treated cells. Analysis of DNA fragmentation in combination with Hoechst 33258 staining indicates that apoptosis is the dominant mode of cell death in the present 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 noted at 2 h after initiation of reincubation and rapidly declined to control 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 protective effect. Maximal protection (85.7 ±5.4%) of the cells from ischemia/reincubation-induced cell damage was achieved at 0.1 p~M BN 50739. The PAF antagonist lacked any protective effect against ischemia-induced cell death. On the other hand, the addition of the stable PAF analogue 1 -O-hexadecyl-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 HN33.1 1 cells with the Ca 2chelator 1,2-b/s (2-aminophenoxy)ethane-N , N, N, N-tetraacetic acid acetoxymethyl ester (BAPTA-AM) also provided a protective effect against ischemia/reincubation-induced cell damage. BAPTA-AM increased cell viability by 50%. Pretreatment with BAPTA-AM also decreased ischemia/reincubation-induced PAF accumulation in HN33.11 cells. The results suggest that PAF, acting via a PAF receptor, is at least in part mediating apoptosis under ischemia/ postischemia-like conditions in HN33.1 1 cells.