Constitutive Release of ATP and Evidence for Major Contribution of Ecto-nucleotide Pyrophosphatase and Nucleoside Diphosphokinase to Extracellular Nucleotide Concentrations

作者：Eduardo Rodolfo Lazarowski, Richard C. Boucher, T. Kendall Harden · 发表于：Journal of Biological Chemistry · 年份：2000 · DOI：10.1074/jbc.m003255200 · 被引用次数：315 · 研究领域：Adenosine and Purinergic Signaling、Biochemical and Molecular Research、Genetics and Neurodevelopmental Disorders

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 (t(1/2);1 approximately;230-40 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-(32)P]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-(32)P]...