Constitutive release of ATP and evidence for major contribution of ecto-nucleotide pyrophosphatase and nucleoside diphosphokinase to extracellular nucleotide concentrations

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 extracel lular nucleotides is well established, the relative flux of ATP and UTP thr ough their extracellular metabolic products remains undefined. Therefore, w e have studied the kinetics of accumulation and metabolism of endogenous AT P in the extracellular medium of four different cell lines. ATP concentrati ons reached a maximum immediately after change of medium and decreased ther eafter with a single exponential decay (t1/2 similar to 30-40 min). ATP lev els did not fall to zero but attained a base-line concentration that was in dependent of the medium volume and of the initial ATP concentration, Althou gh the base line concentration of ATP remained stable for up to 12 h, [gamm a-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-phospha te of ATP was rapidly, although transiently, transferred during steady stat e to species subsequently identified as UTP and GTP, indicating the existen ce of both ecto-nucleoside diphosphokinase activity and the accumulation of endogenous UDP and GDP, Conversely, addition of [gamma-P-32]UTP, resting c ells resulted in transient formation of [gamma-P-32]ATP, indicating phospho rylation of endogenous ADP by nucleoside diphosphokinase, The final P-32-pr oducts 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]inor ganic 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]orthophos phate. These results illustrate for the first time a constitutive release o f ATP and other nucleotides and reveal the existence of a complex extracell ular metabolic pathway for released nucleotides, In addition to the existen ce of an ecto-ATPase activity, our results suggest a major scavenger role o f ecto-ATP pyrophosphatase and a transphosphorylating activity of nucleosid e diphosphokinase.