稀土离子跨人血红细胞膜的荧光法研究

采用Fura-2荧光浓度指示剂对红细胞的稀土跨膜作用进行了系列研究.结果表明,稀土离子不能通过完整的红细胞膜进入细胞内.通过与离子载体实验相对照,发现细胞ATP耗竭后,低浓度的稀土离子(5×10^-6mol/L)不能跨膜进入ATP-耗竭红细胞.KCl去极化及加入电压依赖性钙通道刺激剂Bay-K8644对稀土离子的跨膜也没有促进作用.在Ca²⁺内流正常的情况下,低浓度稀土离子(5×10^-6mol/L)对钙离子内流无影响.增大稀土离子浓度到5×10^-4mol/L,用显微镜观察此时红细胞已开始溶血.在模拟胞内离子组分的缓冲液中(pH=7.05),比较了La³⁺,Eu³⁺和Ca²⁺对Fura-2的敏感程度.此条件下Fura-2对La³⁺和Eu³⁺的检测限分别为10-12和10-14mol/L,对Ca²⁺的检测限为10-8mol/L,并测得Fura-2-La³⁺(Eu³⁺)的络合比为1∶1,表观离解常数为1.7×10^-12和4.95×10^-14mol/L,表明用此法检测稀土离子跨膜行为相当灵敏有效.

Studies on Trans-human Red Cells of Rare Earth Ions by Fura-2 Indicator

The transmembrane behavior of La³⁺ and Eu³⁺ was examined by using Fura-2 technique. The result showing a low concentration La³⁺ or Eu³⁺ (5×10^-6mol/L) could not enter fresh and ATPdepleted human red cells. But in the presence of ionomycin, obvious transmembrane phenomenon was observed when La³⁺ (or Eu³⁺)^of 5×10^-6mol/Lwas added. KCl and Bay K8644 could not promote the influx of La³⁺ (or Eu³⁺). Moreover, a low concentration La³⁺ (or Eu³⁺) had no effect on the influx of Ca ²⁺ in ATPdepleted human red cells. When a higher concentration La³⁺ or Eu³⁺ (5×10^-4mol/L) was added, red cells became hemolysis. Acharacterization of La³⁺ (or Eu³⁺)-Fura-2 interactions showed that they formed a 1∶1 La³⁺ (or Eu³⁺)-Fura-2 complex. In vitro studies confirmed that Fura-2 fluorescence was sensitive to La³⁺ (or Eu³⁺) and the apparent dissociation constants of La³⁺ and Eu³⁺ for Fura-2 (K_d) were 1.7×10^-12 and 4.95×10^-14mol/L, respectively (pH=7.05).