Constitutive release of ATP and evidence for major contribution of ecto-nucleotide pyrophosphatase and nucleoside diphosphokinase to extracellular nucleotide concentrations
Constitutive release of ATP and evidence for major contribution of ecto-nucleotide pyrophosphatase and nucleoside diphosphokinase to extracellular nucleotide concentrations
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DOI：
10.1074/jbc.m003255200
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发表时间：
2000-10-06
影响因子：
4.8
通讯作者：
Harden, TK
中科院分区：
文献类型：
--
作者：
Lazarowski, ER;Boucher, RC;Harden, TK
核苷酸是重要的胞外信号分子，至少有5种哺乳动物的P2Y受体被ATP、UTP、ADP或UDP特异性激活，尽管存在代谢胞外核苷酸的胞外酶，但ATP和UTP通过其胞外代谢产物的相对通量仍未确定。因此，我们研究了四种不同细胞系细胞外培养上清液中内源性三磷酸腺苷的积累和代谢动力学。ATP浓度在换液后即刻达到最大值，之后呈单次指数下降(t1/2类似于30-40min)。ATP水平并没有下降到零，而是达到了一个与培养液体积和初始浓度无关的基线浓度，尽管ATP的基线浓度在长达12 h的时间内保持稳定，但作为放射性示踪剂添加到静止细胞中的[Gamma-P-32]ATP在120min内完全降解，表明稳态反映了ATP水解平衡的基本释放速率(20-200fmolxmin(-1)x cell(-6))。高效液相色谱分析表明，ATP的γ-磷酸在稳态时迅速转移到后来被鉴定为UTP和GTP的物种中，这表明胞外核苷二磷酸激酶活性的存在以及内源UDP和GDP的积累，相反，[Gamma-P-32]UTP的加入，静息细胞导致[Gamma-P-32]ATP的瞬时形成，表明核苷二磷酸激酶对内源ADP的磷酸化。[γ-P-32]ATP代谢的最终P-32产物是[P-32]正磷酸和一种P-32标记物种，进一步纯化并鉴定为[P-32]无机焦磷酸。在C6细胞中，[伽马-P-32]ATP在稳态时生成[P-32]焦磷酸的能力是[P-32]正磷酸的3倍。这些结果首次说明了ATP和其他核苷酸的结构性释放，并揭示了释放的核苷酸存在复杂的细胞外代谢途径，除了胞外-ATPase活性的存在，我们的结果表明外胞-ATP焦磷酸酶和核苷二磷酸激酶的转磷酸化活性是主要的清除剂作用。
Nucleotides are important extracellular signaling molecules, At least five mammalian P2Y receptors exist that are specifically activated by ATP, UTP, ADP, or UDP, Although the existence of ectoenzymes that metabolize extracellular nucleotides is well established, the relative flux of ATP and UTP through their extracellular metabolic products remains undefined. Therefore, we have studied the kinetics of accumulation and metabolism of endogenous ATP in the extracellular medium of four different cell lines. ATP concentrations reached a maximum immediately after change of medium and decreased thereafter with a single exponential decay (t1/2 similar to 30-40 min). ATP levels did not fall to zero but attained a base-line concentration that was independent of the medium volume and of the initial ATP concentration, Although the base line concentration of ATP remained stable for up to 12 h, [gamma-P-32]ATP added to resting cells as a radiotracer was completely degraded within 120 min, indicating that steady state reflected a basal rate of ATP release balanced by ATP hydrolysis (20-200 fmol x min(-1) x cell(-6)). High performance liquid chromatography analysis revealed that the gamma-phosphate of ATP was rapidly, although transiently, transferred during steady state to species subsequently identified as UTP and GTP, indicating the existence of both ecto-nucleoside diphosphokinase activity and the accumulation of endogenous UDP and GDP, Conversely, addition of [gamma-P-32]UTP, resting cells resulted in transient formation of [gamma-P-32]ATP, indicating phosphorylation of endogenous ADP by nucleoside diphosphokinase, The final P-32-products of [gamma-P-32]ATP metabolism were [P-32]orthophosphoric acid and a P-32-labeled species that was further purified and identified as [P-32]inorganic pyrophosphate, In C6 cells, the formation of [P-32]pyrophosphate from [gamma-P-32]ATP at steady state exceeded by 3-fold that of [P-32]orthophosphate. These results illustrate for the first time a constitutive release of ATP and other nucleotides and reveal the existence of a complex extracellular metabolic pathway for released nucleotides, In addition to the existence of an ecto-ATPase activity, our results suggest a major scavenger role of ecto-ATP pyrophosphatase and a transphosphorylating activity of nucleoside diphosphokinase.