eATP is hydrolyzed by the macrophage ectoenzyme CD39. (A-C) The hydrolysis of 500 μM ATP by wild-type BMDMs (closed squares; solid black line) over time compared with RAW264.7 cells (open squares; gray, dashed line) (A), in the presence of 10μM POM-1 (open squares; black, dashed line) (B), or Cd39^−/− (open squares; black, dashed line) BMDMs (C) and was measured by the P_i release assay. (D) The percent of adenine nucleotides hydrolyzed within 30 minures as measured by P_i released by wild-type BMDMs (solid bars) and Cd39^−/− (striped bars) BMDMs in the absence (left) or presence (right) of apyrase. (A-D) The means ± SD of triplicate determinations are shown and are representative of 3 independent experiments. (E) The percent of 5 μM (open squares) or 500 μM (closed squares) ATP hydrolyzed over time by wild-type BMDMs (solid lines) and Cd39^−/− (dashed lines) BMDMs. Percent of hydrolysis was compared with levels of ATP detected in the absence of cells and determined by the ATPlite assay. The data are representative of 3 independent experiments. (F-G) The ³¹P NMR spectra of 500 μM ATP by wild-type (F) or Cd39^−/− BMDMs (G) are shown over time. The location of the α,β,γ phosphorous peaks of ATP (black circles), α,β phosphorous peaks of ADP (gray circles), phosphorous peaks of AMP (white circles), and P_i (black diamonds) was based on reference ³¹P NMR spectra. NMR spectra are representative of at least 3 independent experiments. WT, wild-type.