Involvement of Sodium in Early Phosphatidylserine Exposure and Phospholipid Scrambling Induced by P2X7 Purinoceptor Activation in Thymocytes

Abstract

Extracellular ATP (ATP ec ), a possible effector in thymocyte selection, induces thymocyte death via purinoceptor activation. We show that ATP ec induced cell death by apoptosis, rather than lysis, and early phosphatidylserine (PS) exposure and phospholipid scrambling in a limited thymocyte population (35-40%). PS externalization resulted from the activation of the cationic channel P2X7 (formerly P2Z) receptor and was triggered in all thymocyte subsets although to different proportions in each one. Phospholipid movement was dependent on ATP ec -induced Ca 2؉ and/or Na ؉ influx. At physiological external Na ؉ concentration, without external Ca 2؉ , PS was exposed in all ATP ec -responsive cells. In contrast, without external Na ؉ , physiological external Ca 2؉ concentration promoted a submaximal response. Altogether these data show that Na ؉ influx plays a major role in the rapid PS exposure induced by P2X7 receptor activation in thymocytes. Extracellular ATP (ATP ec )1 induces thymocyte apoptosis and may play a role in thymocyte maturation (1-4). A major change of cell membrane during apoptosis, slightly preceding nuclear condensation (5), is the exposure on the exoplasmic cell surface of phosphatidylserine (PS) (6 -9), a phospholipid normally maintained on the inner membrane leaflet by the aminophospholipid translocase (APLT) (10). This process is associated with a general loss of the asymmetric distribution (scrambling) of all the other major phospholipids (6,7,9). The presence of specific PS receptors on macrophages potentially allows a rapid elimination of apoptotic cells by phagocytosis before their lysis (11). Rapid PS exposure and scrambling are generally observed after platelet activation or treatment of cells with a Ca 2ϩ ionophore, both conditions promoting a drastic and rapid rise in cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] i ) (7). An involvement of Ca 2ϩ has been also reported in PS exposure during apoptosis (6,7,12). Ca 2ϩ acts by inhibiting APLT and by targeting scramblase (6, 7, 13) or other molecular complexes, which could contain phosphoinositides (14,15). Recent studies show that the rapid Ca 2ϩ -induced PS exposure is correlated with scramblase expression (16), in contrast to the delayed PS exposure associated with apoptosis (17, 18), in agreement with the involvement of other phospholipid transporters in apoptotic pathways.ATP ec is able to evoke physiological responses in a wide variety of cells and tissues. ATP is concentrated in cell cytosol and in exocytic vesicles, reaching 2-4 and 100 mM, respectively (19), and can be merely released from damaged cells, transported through carriers or channels, or secreted (19 -21). In the extracellular medium, its lifetime is very short due to its rapid degradation by ectonucleotidases and ecto-ATPases (19). ATP ec binds to distinct types of purinergic receptors classified into two subfamilies: metabotropic P2Y receptors coupled to Gprotein (P2Y1-6 and P2Y11) and ligand-gated ion channel P2X receptors (P2X1-7) (22). P2X7 receptor, previously designat...

P2X7 receptor activation induces reactive oxygen species formation in erythroid cells

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CAY10593 inhibits the human P2X7 receptor independently of phospholipase D1 stimulation

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Cell shrinkage and monovalent cation fluxes: Role in apoptosis

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