Interplay of constitutively released nucleotides, nucleotide metabolism, and activity of P2Y receptors

Abstract

AbstractAt least six mammalian P2Y receptors exist that are specifically activated by ATP, UTP, ADP or UDP. Although the existence of ectoenzymes that rapidly metabolize extracellular nucleotides is well established, the relative flux of ATP and UTP through their extracellular metabolic products remains undefined. In addition, the existence of basal nucleotide release and the contribution of resting levels of ATP and UTP to P2 receptor activation are poorly understood. In the absence of exogenous agonists, an apyrase‐sensitive inositol phosphate accumulation was observed in resting 16HBE14o– human bronchial epithelial cells endogenously expressing P2Y receptors and in 1321N1 human astrocytoma cells expressing a recombinant P2Y2 receptor. To test whether nucleotide release may account for basal P2 receptor activities, the rates of extracellular accumulation and metabolism of endogenous ATP were examined with resting 16HBE14o–, C6 rat glioma, and 1321N1 cell cultures. Although extracellular ATP concentrations (1‐5 nM) remained unchanged for up to 12 h, [γ32P] ATP included in the medium (as a radiotracer) was completely degraded within 120 min, indicating that ATP release balanced ATP hydrolysis. The calculated basal rates of ATP release ranged from 20 to 200 fmol/min per million cells. HPLC analysis during steady state revealed that the gamma‐phosphate of ATP was reversibly transferred to species further identified as UTP and GTP, implicating ecto‐nucleoside diphosphokinase (NDPK)‐catalyzed phosphorylation of endogenous UDP and GDP. At steady state, the final 32P‐products of [γ32P]ATP metabolism were 32P‐orthophosphoric acid and a species further purified and identified as 32P‐pyrophosphate. Constitutive nucleotide release balanced by the concerted activities of ecto‐ATPase, ecto‐ATP pyrophosphatase, and ecto‐NDPK may determine the resting levels of extracellular nucleotides and therefore, the basal activity of P2 receptors. Drug Dev. Res. 53:66–71, 2001. © 2001 Wiley‐Liss, Inc.