QUANTITATIVE CHARACTERIZATION OF HIGH-AFFINITY AND LOW-AFFINITY BINDING-SITES FOR BASIC FIBROBLAST GROWTH-FACTOR ON TRABECULAR CELLS OF THEEYE

By radioligand binding followed by Scatchard analysis, we characterize d and quantitated the specific binding sites for bFGF on cultured trab ecular meshwork cells obtained from freshly enucleated porcine eyes. W e detected two binding sites: 1.67 x 10(4) +/- 5.75 x 10(3) high-affin ity receptors per cell with a K-d of 33.4 +/- 7.90 pM, and 1.70 x 10(6 ) +/- 7.57 x 10(5) low-affinity binding sites per cell with a K-d of 3 .84 +/- 1.41 nm. At low concentrations of I-125-bFGF (< 1.50 ng ml(-1) ), binding was primarily determined by the high-affinity receptors and , at high concentrations (> 2.50 ng ml(-1)), binding was dependent on the low-affinity binding sites. By phase-contrast time-lapse video mic rography and sequential photomicrography, we demonstrated that at a co ncentration of 1 ng ml(-1), bFGF significantly stimulated the rate of mitosis of the trabecular meshwork cells in G(0)-phase compared with c ontrol cultures maintained in serum-free medium alone. Treatment with higher concentrations Of bFGF did not reveal more potent effects on th ese cells. Our findings demonstrate that trabecular meshwork cells do possess low- and high-affinity receptors for bFGF and that bFGF :induc es these cells in vitro to re-enter the cell cycle. Because the low-af finity interactions of I-125-bFGF were reduced by 75% following pretre atment of the trabecular meshwork cells with heparinase, these sites r epresent cell-associated heparin-like molecules and heparan sulfate pr oteoglycans, and may control the bioavailability of bFGF to ocular tis sues. Heparinase treatment also resulted in a 30% reduction in high-af finity binding, which may be, secondary to the decreased low-affinity binding. This finding agrees with the well-established scheme for bFGF -receptor interaction. We conclude that bFGF at the concentration pres ent in aqueous humor is capable of stimulating the mitotic activity of trabecular meshwork cells in vitro, suggesting a possible paracrine r ole of aqueous humour bFGF in vivo. The results obtained in this study , together with our previous findings on bFGF mRNA expression by trabe cular meshwork cells and protein deposition in this tissue, also indic ates that trabecular cells of the eye may utilize bFGF by an autocrine mechanism. (C) 1997 Academic Press Limited.