Free concentrations of intracellular fluorescent anions determined by cytoplasmic dialysis of isolated hepatocytes.
Free concentrations of intracellular fluorescent anions determined by cytoplasmic dialysis of isolated hepatocytes.
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通过分离肝细胞的细胞质透析测定细胞内荧光阴离子的游离浓度。
DOI：
10.1152/ajpgi.1994.267.5.g922
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
Maglova,LM
中科院分区：
文献类型：
--
作者：
Weinman,SA;Maglova,LM
细胞内有机离子存在于与细胞质蛋白质结合的游离溶液中，划分在细胞内膜内，并封闭在细胞内囊泡和细胞器中。本研究的目的是开发一种测量有机离子游离胞质浓度的方法。这是通过测量膜片钳移液管和细胞质之间的初始扩散速率并确定该过程的零点浓度来实现的。使用羧基二甲基荧光素（CF）作为模型化合物。它很容易在细胞质和移液管之间扩散，移液管中的浓度与平衡细胞荧光之间存在线性关系。当先前装载 CF 的细胞被修补时，细胞内荧光迅速向正或负方向变化，具体取决于移液管中 CF 的浓度。零点定义为细胞既不获得也不失去荧光的浓度，描述了游离浓度和总细胞荧光之间的相同关系，与通过直接加载细胞至平衡所确定的关系相同。在预加载荧光胆汁酸衍生物胆酰氨基荧光素 (CGamF) 的肝细胞中，通过暴露 (0.05 µM) 30 分钟，零点出现在移液管中 CGamF 浓度为 0.6 µM 处。该值比浴中的值大12倍。总之，描述了一种可以测量荧光分子游离胞质浓度的新方法。它应该被证明有助于确定运输有机离子的细胞内位置和状态。
Intracellular organic ions exist in free solution bound to cytoplasmic proteins, partitioned within intracellular membranes, and enclosed in intracellular vesicles and organelles. The aim of this study was to develop a method for measurement of the free cytosolic concentration of organic ions. This was accomplished by measuring initial rates of diffusion between patch-clamp pipettes and cell cytoplasm and determining the null-point concentration of this process. Carboxydimethylfluorescein (CF) was used as a model compound. It readily diffused between cytoplasm and pipette, and there was a linear relationship between concentration in the pipette and equilibrium cell fluorescence. When cells previously loaded with CF were patched, intracellular fluorescence rapidly changed in a positive or a negative direction, depending on the concentration of CF in the pipette. The null point, defined as the concentration at which cells neither gained nor lost fluorescence, described the same relationship between free concentration and total cell fluorescence as that determined by direct loading of the cells to equilibrium. In hepatocytes preloaded with a fluorescent bile acid derivative, cholylglycylamidofluorescein (CGamF), by exposure (0.05 microM) for 30 min, the null point occurred at a CGamF concentration in the pipette of 0.6 microM. This value is 12 times greater than that in the bath. In conclusion, a new method is described that can measure free cytosolic concentrations of fluorescent molecules. It should prove useful in determining the intracellular location and state of transported organic ions.