Characterization of platelet-activating factor binding to human airway epithelial cells: Modulation by fatty acids and ion-channel blockers

Abstract

Radioligand-binding studies were performed in primary cultured human airway epithelial cells with [3H]PAF to determine whether these cells express platelet-activating factor (PAF) receptors. Scatchard analysis of PAF binding data revealed a single class of PAF binding sites with K(d) 1.8 ± 0.2 nM and B(max) 21.0 ± 2.1 fmol/ 106 cells (13000 receptors/cell). PAF binding increased the intracellular free Ca2+ concentration ([Ca2+](i)), indicating functional PAF receptors. Palmitate (C(16:0)), linoleic acid (C(18:2ω6) or eicosapentaenoic acid (C(20:5ω3)) was incubated with the cells to test the effect on PAF binding. Incorporation of each fatty acid into cellular phospholipid occurred. [3H]PAF (1 nM) binding decreased in cells supplemented with C(20:5ω3) but increased in the cells supplemented with C(16:0) Scatchard analysis revealed that the inhibition of PAF binding by supplementation with C(20:5ω3) was due to a decrease in both affinity and number of PAF receptors. PAF-stimulated increase in [Ca2+](i) was also decreased by 60% in cells supplemented with C(20:5ω3). Verapamil, a Ca2+-channel blocker, and amiloride, a Na+-channel blocker, inhibited specific binding of [3H]PAF to the cells, with IC50 4-5 μM and 0.2 mM respectively. Diphenylamine-2-carboxylate (DPC), a Cl--channel blocker, dramatically increased PAF binding to the cell in a dose-dependent manner. Scatchard analysis revealed that verapamil and amiloride decreased both binding affinity and number of PAF receptors, whereas DPC increased PAF binding sites without affecting binding affinity. These results demonstrate that human airway epithelial cells have a functional receptor for PAF and that PAF receptor binding can be modulated by exogenous fatty acids and by ion-channel blockers.