Apoptosis of murine thymocytes induced by extracellular ATP is dose- and cytosolic pH-dependent

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Thymocytes from young Balb/C mice responded to low extracellular ATP (ATP ec) doses (≤0.3 mM) with a rapid intracellular acidification (mean pH: ca. 0.3 pH unit) that was inhibited by the Ca 2+ channel blocker verapamil, or by suramin (50 μM) and TNP-ATP (40 μM), potent P2x (and P2y) purinoreceptor antagonists. ATP ec also triggered a remarkable DNA fragmentation and cell shrinkage detectable only at these low doses. DNA fragmentation gradually disappears with increasing [ATP ec] above 0.5 mM, with a concomitant dominance of cytosolic alkalinization of the cells. Suramin and TNP-ATP also blocked the ATP ec-triggered DNA fragmentation efficiently. oATP, inhibitor of P2z nonspecific ATP-gated membrane pores, and 2 mM extracellular Mg 2+ did not influence either the cytosolic acidification or the DNA fragmentation, but almost completely abolished the intracellular alkalinization characteristic of P2z receptor activation at high ATP ec doses. Antagonist-sensitivity of the ATP ec-induced membrane potential responses indicates that hyperpolarization is associated with intracellular acidification, while rapid depolarization is linked to alkalinization. These data together indicate that the Ca 2+-dependent hyperpolarization and cytosolic acidification triggered by low ATP ec doses are essential early signals in apoptosis of murine thymocytes and are likely mediated by P2x1 type ATP-gated ion channels. Subset specificity of the early purinergic signals suggests that the double positive thymocytes are most sensitive to ATP ec showing both P2z and P2x receptor activation characteristics, the double negative thymocytes preferentially show P2z-type, while single positive (CD4 −CD8 + or CD4 +CD8 −) thymocytes respond mostly by weaker P2x-type changes, indicating that ATP ec, similarly to adenosine may serve as a potential regulator of cell death and differentiation in the thymus.