Fiedler J L, Pollard H B, Rojas E
Laboratory of Cell Biology and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
J Membr Biol. 1992 Apr;127(1):21-33. doi: 10.1007/BF00232755.
We and others have shown previously that ATP is secreted from mouse brain synaptosomes following depolarization of the membrane by high [K+]o and the time course can be monitored accurately by measuring the light emitted from luciferin-luciferase included in the reaction medium. In the present work we have evaluated the relative importance of [Ca2+]o and membrane potential on the ATP secretion process by modelling the time course of ATP release under different conditions. After correction of the records for destruction of released ATP by synaptosomal ecto-ATPase activity, we found that ATP secretion occurs by an apparent first order process. We also established that, in addition to the classical [Ca2+]o-dependent mode, ATP secretion also occurred in the absence of extracellular calcium ([Ca2+]o less than 1 microM). Upon lowering the extracellular Ca2+ concentration, both the rate and the extent of ATP secretion decreased. To assess the contribution of membrane potential to the release rate we measured ATP secretion at membrane potentials determined by extracellular [K+]o (or [Rb+]o) as defined by the distribution of the carbocyanine dye, diSC3(5). Rate constants computed from measured secretion curves revealed that this parameter was essentially independent of membrane potential in the absence of [Ca2+]o. Noise analysis of the light signal showed that the variance increased upon stimulation by high [K+]o, suggesting that both modes of secretion are quantal. Thus, we conclude that the rate of ATP secretion from nerve terminals depends upon Ca2+ entry but not on membrane potential, per se.
我们和其他研究人员之前已经表明，在高[K⁺]ₒ使细胞膜去极化后，小鼠脑突触体可分泌ATP，并且通过测量反应介质中荧光素 - 荧光素酶发出的光，可以准确监测其时间进程。在本研究中，我们通过模拟不同条件下ATP释放的时间进程，评估了[Ca²⁺]ₒ和膜电位对ATP分泌过程的相对重要性。在用突触体胞外ATP酶活性校正释放的ATP被破坏的记录后，我们发现ATP分泌通过一个明显的一级过程发生。我们还确定，除了经典的[Ca²⁺]ₒ依赖性模式外，在没有细胞外钙（[Ca²⁺]ₒ小于1微摩尔）的情况下也会发生ATP分泌。降低细胞外Ca²⁺浓度时，ATP分泌的速率和程度均下降。为了评估膜电位对释放速率的贡献，我们在由细胞外[K⁺]ₒ（或[Rb⁺]ₒ）决定的膜电位下测量ATP分泌，膜电位由羰花青染料diSC₃(5)的分布定义。从测量的分泌曲线计算出的速率常数表明，在没有[Ca²⁺]ₒ的情况下，该参数基本上与膜电位无关。光信号的噪声分析表明，在高[K⁺]ₒ刺激下方差增加，这表明两种分泌模式都是量子化的。因此，我们得出结论，神经末梢ATP分泌的速率取决于Ca²⁺内流，而本身不取决于膜电位。
