POSSIBLE EXISTENCE OF 2 SUBSETS OF PLATELET-ACTIVATING-FACTOR RECEPTOR TO MEDIATE POLYPHOSPHOINOSITIDE BREAKDOWN AND CALCIUM INFLUX IN NEUROBLASTOMA X GLIOMA HYBRID NG-108-15 CELLS

Platelet-activating factor (PAF) initiated polyphosphoinositide (PolyPI) breakdown and a rise of intracellular calcium concentration ([Ca2+]i) in neuroblastoma x glioma hybrid NG 108-15 cells. The accumulation of [H-3]inositol trisphosphate and [H-3]inositol bisphosphate was evident within 15 s after PAF stimulation, peaked at 1 min, and then gradually decayed. The increase in [H-3]inositol monophosphate level was observed at 30 s, plateaued in 5 min, and was sustained up to 10 min in the presence of 10 mM LiCl. On the other hand, the rise of [Ca2+]i evoked by PAF reached a peak within 8-12 s and returned to basal levels within 1 min as measured in fura 2-loaded cells. When cells were suspended in Ca2+-depleted medium, the PAF-induced [Ca2+]i rise was reduced by 80%, indicating that the increase of [Ca2+]i was predominantly due to the Ca2+ influx from an extracellular source. Both PAF-induced accumulation of H-3-labeled inositol phosphates and [Ca2+]i elevation were concentration dependent with EC50 values of approximately 1 x 10(-10) and 5 x 10(-8), respectively. The PAF analogs 1-O-hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine and 1-O-hexadecyl-2-O-methyl-rac-glycerol-3-phosphocholine were much poorer agonists at eliciting the same responses in these cells. Pretreatment of cells with pertussis toxin caused a substantial inhibition of PAF-induced accumulation of H-3-inositol phosphates. In contrast. the rise in [Ca2+]i was not significantly affected by toxin treatment at the same concentration. SRI-63675, a PAF antagonist, blocked both PAF-induced responses effectively, whereas another antagonist, BN-52021, at the same dose inhibited substantially the accumulation of H-3-inositol phosphates but had a negligible effect on the Ca2+ response. Scatchard analysis of radioligand saturation binding data revealed the existence of two PAF binding sites in NG 108-15 cell membranes: one was high affinity with a dissociation constant (K(D)) of 6.1 +/- 1.8 x 10(-11) M and a receptor density (B(max)) of 54.0 +/- 2.8 fmol/mg of protein; another was low affinity with a K(D) of 1.7 +/- 0.4 x 10(-8) M and a B(max). of 5.6 +/- 0.5 pmol/mg of protein. Displacement of specific [H-3]PAF binding by unlabeled PAF also demonstrated two binding sites with inhibitory constant (K(i)) values of 7.5 +/- 2.5 x 10(-11) and 2.7 +/- 1.7 x 10(-7) M, respectively. BN-52021 and SRI-63675 displayed differential affinities to these two binding sites. Taken together, these data suggest the existence of two subtypes of PAF receptor to mediate polyPI hydrolysis and Ca2+ influx in NG 108-15 cells.