Fig. 7.

Increased ecto-AMPase activity of RIP-deficient Jurkat cells is not correlated with up-regulation of ecto-AMP deaminase. (A) Schematic of extracellular pathways, ectoenzymes (green), and inhibitors (red) that can mediate the metabolism/clearance of released AMP. (B) We added 100 μM AMP minus (B-i, upper traces) or plus (B-ii, lower traces) 50 μM dipyridamole to WT or RIP1-deficient cells (2 × 10⁶ cells/ml in BSS + 5 mM glucose + 0.1% BSA). Cell-free supernatants were collected after 30 minutes and processed for separation and quantification of AMP, IMP, adenosine, and inosine by reverse phase HPLC and absorbance detection. Arrows indicate elution times of AMP, IMP, adenosine, and inosine standards; ? indicates an unidentified peak. Data are representative of four to six experiments. (C) WT or RIP1-deficient Jurkat cells were treated without or with STS for 4 hours in the presence or absence of 50 μM APCP, 200 μM pentostatin, or combined APCP plus pentostatin. Data indicate mean ±S.E. of three independent experiments. Analysis by two-way analysis of variance and Tukey post-test comparison; a: +STS versus −STS; b:RIP1-def versus WT; c: +STS with pentostatin or pentostatin plus APCP versus STS alone. All panels: ns, not statistically significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.