淋巴细胞膜上Na^(+)/Ca^(2+)交换操纵的Eu^(3+)内流的荧光法研究

Studies on Eu^(3+) Influx Mediated by the Na^(+)/Ca^(2+) Exchanger in Human Lymphocytes by the Fluorescent Method

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Eu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Ca2+通道阻断剂nicardipine对Eu3+的进入无显著影响,建议了Na+/Ca2+交换是Eu3+进入细胞的主要途径。当加入不同浓度的Eu3+和Ca2+的混和组分时,在无Na+介质中测试,结果表明Eu3+与Ca2+竞争Na+/Ca2+交换位点。在模拟胞内离子组分的EGTA缓冲液中(pH7.05),测得Eu3+与Fura-2的离解常数为4.95×10-14mol·L-1。

We examined whether Eu 3? can entry human peripheral blood lym phocytes by the Na ?/Ca 2? exchange using fura-2technique by measuring the quenching of fluorescence at ￡3￡?￡°nm excitation￡(r)The results showed that the pretreatmemt of lymphocytes for ￡3￡°m in at ￡3￡*??with ￡°￡(r)1mmol·L-1 ouabain and followed by exposure to d ifferent concentrations of Eu3?in Na ?-free medium resulted in a decrease i n the 360nm fluorescence intensity,and intra-cellular Eu 3?concentrations were directly proportional to the extracellular Eu3?concentrations and were founded to be ￡2￡(r)76×10-14mol?¤L￡-￡± in cells exposured to ￡°￡(r)4mmol·L -1 Eu 3? ￡(r)Eu 3? influx was entirely dependent on ouabain pretreatment and reversal o f the Na ? gradients as expected for Na ? /Eu 3? exchange activity￡(r)No further effect on the 360nm signal was seen wi th nicardipine pretreated,we conclude that the Na ? /Ca 2? exchanger pro-vides the major route of Eu 3? entry in human lymphocytes￡(r)Furthermore,when composite of different concentrations of Eu 3? and Ca 2?in the extracellular Na ? -free medium was varied,the Eu 3? influx significantly decreased as t he extracellular Ca 2? concentration increased,it is suggested that the Ca 2? influx and Eu 3? influx by the Na ? /Ca 2? exchange￡-mediated might be mutuall y competition￡(r)A characterization o f Eu-fura-2interactions in solutio n simu-lating intracellular ionic composition showed that Eu 3? forms a ￡±??￡±Eu￡-fura￡-￡2complex￡(r)In vit ro studies confirmed that fura-2fluorescence was sensit ive to Eu 3? and the apparent dissociation constant of Eu 3? for fura￡-￡2￡¨K d ￡(c)was￡′￡(r)95×10 -14 mol·L ￡-￡± ￡¨pH ￡*￡(r)￡°￡μ￡(c)￡(r)