Sierra E E, Brigle K E, Spinella M J, Goldman I D
Department of Medicine, Virginia Commonwealth University, Medical College of Virginia, Richmond 23298, USA.
Biochem Pharmacol. 1995 Oct 12;50(8):1287-94. doi: 10.1016/0006-2952(95)94097-y.
This laboratory previously described an L1210 murine leukemia cell line with a functional defect in the reduced folate carrier and increased expression of folate receptor-beta (F2-MTXrA). This cell line was used to characterize methotrexate (MTX) influx mediated by folate receptor-beta and to compare this with influx mediated by the reduced folate carrier in L1210 parental cells. Influx of 0.2 microM MTX in F2-MTXrA cells was one-third that of L1210 cells and was abolished by very low concentrations of folic acid. Kinetic analysis revealed that MTX transport mediated by folate receptor-beta exhibited an influx kappa t one-third, and an influx Vmax one-fourth, that of the reduced folate carrier. Metabolic inhibitors markedly suppressed influx in F2-MTXrA cells but had no effect on MTX influx in L1210 cells. MTX influx in both cell lines was inhibited by the organic anions probenecid, sulfobromophthalein, and CI-920, but to a lesser extent in F2-MTXrA cells. The inhibitory effects of these anions on transport in F2-MTXrA cells could be attributed to their inhibition of MTX binding to the folate receptor. Although MTX influx in both cell lines was not sodium dependent, removal of extracellular chloride increased influx 2-fold in L1210 cells while markedly inhibiting influx in F2-MTXrA cells. Substitution of Cl- with isethionate or NO3- partially restored influx in the latter cells, whereas SO4(2-) was inhibitory. Anions enhanced MTX binding to folate receptor-beta with isethionate > SO4(2-) > Cl-. Decreasing the buffer pH to 6.2 produced a 69% reduction, and a 260% increase, in MTX influx in L1210 cells and F2-MTXrA cells, respectively. The data indicate that folate receptor-beta-mediated MTX influx has properties fundamentally different from transport mediated by the reduced folate carrier in terms of energy, ion, and pH dependence. There was no evidence indicating that these processes are functionally linked.
本实验室先前描述了一种L1210小鼠白血病细胞系，其在还原型叶酸载体上存在功能缺陷，且叶酸受体β（F2-MTXrA）表达增加。该细胞系用于表征由叶酸受体β介导的甲氨蝶呤（MTX）内流，并将其与L1210亲本细胞中由还原型叶酸载体介导的内流进行比较。F2-MTXrA细胞中0.2微摩尔MTX的内流是L1210细胞的三分之一，并且被极低浓度的叶酸所消除。动力学分析表明，由叶酸受体β介导的MTX转运表现出的内流κt为还原型叶酸载体的三分之一，内流Vmax为四分之一。代谢抑制剂显著抑制F2-MTXrA细胞中的内流，但对L1210细胞中的MTX内流没有影响。两种细胞系中的MTX内流均受到有机阴离子丙磺舒、磺溴酞钠和CI-920的抑制，但在F2-MTXrA细胞中的抑制程度较小。这些阴离子对F2-MTXrA细胞中转运的抑制作用可归因于它们对MTX与叶酸受体结合的抑制。尽管两种细胞系中的MTX内流均不依赖于钠，但去除细胞外氯离子会使L1210细胞中的内流增加2倍，而显著抑制F2-MTXrA细胞中的内流。用羟乙基磺酸或硝酸根替代氯离子可部分恢复后一种细胞中的内流，而硫酸根则具有抑制作用。阴离子增强MTX与叶酸受体β的结合，其作用顺序为羟乙基磺酸＞硫酸根＞氯离子。将缓冲液pH降至6.2分别使L1210细胞和F2-MTXrA细胞中的MTX内流减少69%和增加260%。数据表明，在能量、离子和pH依赖性方面，叶酸受体β介导的MTX内流具有与还原型叶酸载体介导的转运根本不同的特性。没有证据表明这些过程在功能上是相关联的。
