PAF binding to a single receptor in corneal epithelium plasma membrane

PURPOSE. To study the binding characteristics and the expression of platele t-activating factor receptors (PAF-R) in corneal epithelium to elucidate th e site of action of PAF. METHODS. Binding of [H-3]PAF was investigated in subcellular fractions of t he epithelia of bovine corneas and in membranes from cultured rabbit cornea l epithelial cells, Dose-response inhibition curves of [H-3]PAF-specific bi nding were generated using increasing concentrations of several. PAF-R anta gonists. RNA from rabbit, corneal epithelial cells was probed for PAF-R exp ression by reverse transcription-polymerase chain reaction (RT-PCR) with sp ecifically designed degenerated primers. RESULTS. Scatchard analysis showed a high-affinity binding site in bovine a nd rabbit corneal epithelium. The dissociation constant (K-d) and the maxim um binding sites (B-max) in a bovine membrane preparation and similar rabbi t frac tion were 0.77 +/- 0.03 nM and 180 +/- 21 femtomoles/mg protein and 4.3 nM and 1.3 picomoles/mg protein, respectively. Specific PAF-binding sit es were found in bovine preparations enriched in plasma membranes with a K- d = 69.6 pM and B-max = 80 femtomoles/mg protein; no specific binding was f ound in nuclei or microsomal fractions. RT-PCR of rabbit: corneal epitheliu m generated a single product of the predicted size (478 bp). The deduced am ino acid sequence of the purified PCR product was 87% homologous to human P AF-R. The hetrazepines BN 50727 and BN 50730 and the PAF structural analogu es CV 3988 and CV 6209 competitively inhibited [H-3]PAF binding to corneal epithelium with similar potency. WEB 2086 BS was two orders of magnitude le ss active in antagonizing PAF binding. CONCLUSIONS. Corneal epithelium contains a single population of receptors l ocalized in the plasma membrane. PAF antagonists exert their actions by blo cking this PAF-R. The partial sequence deduced ih rabbit corneal PAF-R show a higher homology to the human PAF-R.