Tripathi R C, Borisuth N S, Li J, Tripathi A J, Tripathi B J
Department of Ophthalmology, University of South Carolina School of Medicine, Columbia 29203, USA.
Exp Eye Res. 1997 Mar;64(3):335-41. doi: 10.1006/exer.1996.0215.
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.
通过放射性配体结合及随后的Scatchard分析，我们对从新鲜摘除的猪眼中获取的培养小梁网细胞上碱性成纤维细胞生长因子（bFGF）的特异性结合位点进行了表征和定量。我们检测到两个结合位点：每个细胞有1.67×10⁴±5.75×10²个高亲和力受体，解离常数（Kd）为33.4±7.90皮摩尔；每个细胞有1.70×10⁴±7.57×10⁵个低亲和力结合位点，Kd为3.84±1.41纳摩尔。在低浓度的¹²⁵I - bFGF（<1.50纳克/毫升）时，结合主要由高亲和力受体决定；在高浓度（>2.50纳克/毫升）时，结合则依赖于低亲和力结合位点。通过相差延时视频显微术和连续显微摄影，我们证明在浓度为1纳克/毫升时，与仅在无血清培养基中培养的对照细胞相比，bFGF显著刺激了处于G0期的小梁网细胞的有丝分裂速率。用更高浓度的bFGF处理并未显示对这些细胞有更强的作用。我们的研究结果表明小梁网细胞确实拥有bFGF的低亲和力和高亲和力受体，并且bFGF在体外可诱导这些细胞重新进入细胞周期。由于在用肝素酶预处理小梁网细胞后，¹²⁵I - bFGF的低亲和力相互作用降低了75%，这些位点代表细胞相关的类肝素分子和硫酸乙酰肝素蛋白聚糖，并且可能控制bFGF对眼组织的生物利用度。肝素酶处理还导致高亲和力结合降低了30%，这可能是低亲和力结合减少的继发结果。这一发现与已确立的bFGF - 受体相互作用模式相符。我们得出结论，房水中存在的浓度的bFGF能够在体外刺激小梁网细胞的有丝分裂活性，提示房水bFGF在体内可能具有旁分泌作用。本研究获得的结果，连同我们先前关于小梁网细胞bFGF mRNA表达及该组织中蛋白质沉积的研究结果，也表明眼的小梁细胞可能通过自分泌机制利用bFGF。
