Characterization of the N-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 N-etheno-bridged adenine nucleotides to evaluate ecto-nucleotidase activity. We observed that the metabolism of N-etheno-ATP versus ATP was quantitatively similar when incubated with recombinant CD39, ENTPD2, ENTPD3, or ENPP-1, and the quantitative metabolism of N-etheno-AMP versus AMP was similar when incubated with recombinant CD73. This suggests that ecto-nucleotidases process N-etheno-bridged adenine nucleotides similarly to endogenous adenine nucleotides. Four cell types rapidly (t, 0.21 to 0.66 h) metabolized N-etheno-ATP. Applied N-etheno-ATP was recovered in the medium as N-etheno-ADP, N-etheno-AMP, N-etheno-adenosine, and surprisingly N-etheno-adenine; intracellular N-etheno compounds were undetectable. This suggests minimal cellular uptake, intracellular metabolism, or deamination of these compounds. N-etheno-ATP, N-etheno-ADP, N-etheno-AMP, N-etheno-adenosine, and N-etheno-adenine had little affinity for recombinant A, A, or A receptors, for a subset of P2X receptors (H-α,β-methylene-ATP binding to rat bladder membranes), or for a subset of P2Y receptors (S-ATP-αS binding to rat brain membranes), suggesting minimal pharmacological activity. N-etheno-adenosine was partially converted to N-etheno-adenine in four different cell types; this was blocked by purine nucleoside phosphorylase (PNPase) inhibition. Intravenous N-etheno-ATP was quickly metabolized, with N-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 N-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 N-etheno-adenosine to N-etheno-adenine.