Abstract
The ATP-gated P2X(7) receptor is a plasma membrane receptor belonging to the family of P2X purinoceptors. Its activation leads to multiple downstream events including influx of ions, pore formation to allow the passage of larger molecular weight species, and cell death by apoptosis and/or necrosis. The cell death is thought to be correlated with the pore formation but does not directly result from the dilatation of pores. We have generated and characterized a clone of chicken DT40 lymphocytes stably transfected with the rat P2X(7) receptor. In this study, we investigated the mechanism of P2X(7) receptor-induced cell death using this clone. Treatment with P2X(7) receptor agonist, 2'-3'-O-(4-benzoylbenzoyl)-ATP induced depolarization of membrane potential, pore formation, and cell shrinkage, an early hallmark of apoptosis in the buffer containing physiological concentrations of ions. Analysis by flow cytometry revealed that the activity of pore formation in shrunk cells was much higher than in non-shrunk cells. The activation of P2X(7) receptor also caused the release of lactate dehydrogenase from cells. The P2X(7) receptor-mediated cell shrinkage and lactate dehydrogenase release were blocked when media Cl(-) was replaced with gluconate. However, removal of extracellular Cl(-) did not affect plasma membrane depolarization and pore formation by treatment with 2'-3'-O-(4-benzoylbenzoyl)-ATP. Therefore we concluded that pore formation plays a critical role in the P2X(7) receptor-induced apoptotic cell death and that this is mediated by extracellular Cl(-) influx.
Highlights
Extracellular ATP plays an important role in cell signaling, modulation of cell growth, differentiation, and induction of cell death by apoptosis and/or necrosis [1, 2]
Apoptotic Cell Death During P2X7 Receptor Activation—The activation of P2X7 receptor initially leads to the opening of ionic channels, which in turn results in the secondary opening of membrane pores that allow the passage of larger molecular species [5]
This cell shrinkage was completely prevented in DMEM/F-12 supplemented with 5 mM MgCl2 (Fig. 2A) that is known to block the activation of P2X7 receptor
Summary
BzATP, 2Ј-3Ј-O-(4-benzoylbenzoyl)ATP; DIDS, 4,4Ј-diisothiocyanatostilbene-2,2Ј-disulfonic acid; DMEM/ F-12, Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 Ham; DiBAC4(3), bis(1,3-dibutylbarbituric acid)-trimethine oxonol; HBSS, Hanks’ based salt solution; LDH, lactate dehydrogenase. Cell shrinkage or loss of cell volume has traditionally been viewed as a passive event during apoptosis, recent studies from several laboratories have shown that cell shrinkage or flux of ions associated with the change in cell size plays a critical role in the regulation of the cell death machinery [18]. The cell shrinkage and its significance in the cell death mediated by P2X7 receptor have not been reported. To understand the molecular mechanism by which P2X7 receptor-induced cell death occurs, we investigated the essential role of pore formation in the apoptotic cell death induced by the activation of. P2X7 receptor in DT40/P2X7 cells and its ionic dependence and selectivity by measuring cell size, an early distinction between apoptosis and necrosis, with a flow cytometer
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