无机化学学报

淋巴细胞膜上Na⁺/Ca²⁺交换操纵的Eu³⁺内流的荧光法研究

Studies on Eu³⁺ Influx Mediated by the Na⁺/Ca²⁺ Exchanger in Human Lymphocytes by the Fluorescent Method

作者	单位
魏春英	山西大学分子科学研究所,太原 030006
杨频	山西大学分子科学研究所,太原 030006

摘要: 利用Fura-2荧光浓度指示剂法、通过检测360nm激发荧光强度的变化,研究了Eu³⁺能否利用人外周血淋巴细胞膜上的Na⁺/Ca²⁺交换进入细胞。结果表明:用ouabain预处理细胞无Na⁺介质中测试,当加入Eu³⁺时,360nm荧光强度发生猝灭,且随着胞外加入的Eu³⁺浓度的增大而猝灭增强。表明在实验条件下Eu³⁺可以进入细胞。电压依赖性Ca

关键词: 淋巴细胞 Na⁺/Ca²⁺交换稀土离子 Fura-2

基金项目:

Abstract: We examined whether Eu³⁺ can entry human peripheral blood lymphocytes by the Na⁺/Ca²⁺ exchange using fura-2 technique by measuring the quenching of fluorescence at 360nm excitation.The results showed that the pretreatmemt of lymphocytes for 30min at 37℃ with 0.1mmol·L^-1 ouabain and followed by exposure to different concentrations of Eu³⁺ in Na⁺-free medium resulted in a decrease in the 360nm fluorescence intensity, and intra-cellular Eu³⁺ concentrations were directly proportional to the extracellular Eu³⁺ concentrations and were founded to be 2.76×10^-14mol·L^-1 in cells exposured to 0.4mmol·L^-1 Eu³⁺. Eu³⁺ influx was entirely dependent on ouabain pretreatment and reversal of the Na⁺ gradients as expected for Na⁺/Eu³⁺ exchange activity.No further effect on the 360nm signal was seen with nicardipine pretreated, we conclude that the Na⁺/Ca²⁺ exchanger pro-vides the major route of Eu³⁺ entry in human lymphocytes. Furthermore, when composite of different concentrations of Eu³⁺ and Ca²⁺ in the extracellular Na⁺-free medium was varied, the Eu³⁺ influx significantly decreased as the extracellular Ca²⁺ concentration increased, it is suggested that the Ca²⁺ influx and Eu³⁺ influx by the Na+/Ca²⁺ exchange-mediated might be mutually competition. A characterization of Eu-fura-2 interactions in solution simu-lating intracellular ionic composition showed that Eu³⁺ forms a 1∶1 Eu-fura-2 complex. In vitro studies confirmed that fura-2 fluorescence was sensitive to Eu³⁺ and the apparent dissociation constant of Eu³⁺ for fura-2 (Kd) was 4.95×10^-14mol·L^-1 (pH 7.05).

Keywords: lymphocytes Na⁺/Ca²⁺ exchange rare earth ions fura-2

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魏春英,杨频.淋巴细胞膜上Na⁺/Ca²⁺交换操纵的Eu³⁺内流的荧光法研究[J].无机化学学报,2002,18(6):571-576.

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