Quantitative characterization of high- and low-affinity binding sites for basic fibroblast growth factor on trabecular cells of the eye.
By radioligand binding followed by Scatchard analysis, we characterized and quantitated the specific binding sites for bFGF on cultured trabecular meshwork cells obtained from freshly enucleated porcine eyes. We detected two binding sites: 1.67 x 10(4) +/- 5.75 x 10(2) high-affinity receptors per cell with a Kd of 33.4 +/- 7.90 pM, and 1.70 x 10(4) +/- 7.57 x 10(5) low-affinity binding sites per cell with a Kd of 3.84 +/- 1.41 nM. At low concentrations of 125I-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 micrography and sequential photomicrography, we demonstrated that at a concentration of 1 ng ml-1, bFGF significantly stimulated the rate of mitosis of the trabecular meshwork cells in G0-phase compared with control cultures maintained in serum-free medium alone. Treatment with higher concentrations of bFGF did not reveal more potent effects on these cells. Our findings demonstrate that trabecular meshwork cells do possess low- and high-affinity receptors for bFGF and that bFGF induces these cells in vitro to re-enter the cell cycle. Because the low-affinity interactions of 125I-bFGF were reduced by 75% following pretreatment of the trabecular meshwork cells with heparinase, these sites represent cell-associated heparin-like molecules and heparan sulfate proteoglycans, and may control the bioavailability of bFGF to ocular tissues. Heparinase treatment also resulted in a 30% reduction in high-affinity 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 present in aqueous humor is capable of stimulating the mitotic activity of trabecular meshwork cells in vitro, suggesting a possible paracrine role of aqueous humour bFGF in vivo. The results obtained in this study, together with our previous findings on bFGF mRNA expression by trabecular meshwork cells and protein deposition in this tissue, also indicates that trabecular cells of the eye may utilize bFGF by an autocrine mechanism.
Tripathi
R C
RC
Department of Ophthalmology, University of South Carolina School of Medicine, Columbia 29203, USA.
Borisuth
N S
NS
Li
J
J
Tripathi
A J
AJ
Tripathi
B J
BJ
D000818:Animals; D016222:Fibroblast Growth Factor 2; D006494:Heparin Antagonists / Q000494:pharmacology; D019763:Heparin Lyase; D008938:Mitosis; D011133:Polysaccharide-Lyases / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011869:Radioligand Assay; D017468:Receptors, Fibroblast Growth Factor / Q000032:analysis*; D013552:Swine; D014129:Trabecular Meshwork / Q000737:chemistry* / Q000166:cytology
D006494:Heparin Antagonists; D017468:Receptors, Fibroblast Growth Factor; D016222:Fibroblast Growth Factor 2; D011133:Polysaccharide-Lyases; D019763:Heparin Lyase