Characterization of the N6-etheno-bridge method to assess extracellular metabolism of adenine nucleotides: detection of a possible role for purine nucleoside phosphorylase in adenosine metabolism

The goal of this study was to determine the validity of using N6-etheno-bridged adenine nucleotides to evaluate ecto-nucleotidase activity. We observed that the metabolism of N6-etheno-ATP versus ATP was quantitatively similar when incubated with recombinant CD39, ENTPD2, ENTPD3, or ENPP-1, and the quantitative metabolism of N6-etheno-AMP versus AMP was similar when incubated with recombinant CD73. This suggests that ecto-nucleotidases process N6-etheno-bridged adenine nucleotides similarly to endogenous adenine nucleotides. Four cell types rapidly (t1/2, 0.21 to 0.66 h) metabolized N6-etheno-ATP. Applied N6-etheno-ATP was recovered in the medium as N6-etheno-ADP, N6-etheno-AMP, N6-etheno-adenosine, and surprisingly N6-etheno-adenine; intracellular N6-etheno compounds were undetectable. This suggests minimal cellular uptake, intracellular metabolism, or deamination of these compounds. N6-etheno-ATP, N6-etheno-ADP, N6-etheno-AMP, N6-etheno-adenosine, and N6-etheno-adenine had little affinity for recombinant A1, A2A, or A2B receptors, for a subset of P2X receptors (3H-α,β-methylene-ATP binding to rat bladder membranes), or for a subset of P2Y receptors (35S-ATP-αS binding to rat brain membranes), suggesting minimal pharmacological activity. N6-etheno-adenosine was partially converted to N6-etheno-adenine in four different cell types; this was blocked by purine nucleoside phosphorylase (PNPase) inhibition. Intravenous N6-etheno-ATP was quickly metabolized, with N6-etheno-adenine being the main product in naïve rats, but not in rats pretreated with a PNPase inhibitor. PNPase inhibition reduced the urinary excretion of endogenous adenine and attenuated the conversion of exogenous adenosine to adenine in the renal cortex. The N6-etheno-bridge method is a valid technique to assess extracellular metabolism of adenine nucleotides by ecto-nucleotidases. Also, rats express an enzyme with PNPase-like activity that metabolizes N6-etheno-adenosine to N6-etheno-adenine.