Abstract
The P2X(7) receptor is known for the cytotoxic activity because of its ability to cause opening of non-selective pores in the plasma membrane and activate apoptotic caspases. A key factor of P2X(7)-dependent cytotoxicity is the massive intracellular Ca(2+) increase triggered by its activation. Here we show that P2X(7) transfection increased the ability of the endoplasmic reticulum to accumulate, store, and release Ca(2+). This caused a larger agonist-stimulated increase in cytosol and mitochondrial Ca(2+) in P2X(7) transfectants than in mock transfected cells. P2X(7) transfectants survived and even proliferated in serum-free conditions and were resistant to apoptosis triggered by ceramide, staurosporin, or intracellular Zn(2+) chelation. Finally, the nuclear factor of activated T cells complex 1 (NFATc1) was strongly activated in the P2X(7) transfectants. These observations support our previous finding that the P2X(7) receptor under tonic conditions of stimulation, i.e. those observed in response to basal ATP release, has an anti-apoptotic or even growth promoting rather than cytotoxic activity.
Highlights
Plasma membrane blebbing [2, 3], rapid release of interleukin-1 via microvesicle shedding [2, 4], cell fusion [5], lymphoid cell proliferation [6], cell death [7, 8], and bone formation/ resorption [9]
endoplasmic reticulum (ER) Ca2ϩ can be transferred to mitochondria at dynamic sites of contact likely controlled by plasma membrane receptors, second messengers, or the metabolic state of the cell [27]
In a previous study we showed that HEK293 cells transfected with the P2X7 receptor (P2X7R) (HEK293-P2X7) have a higher resting mitochondrial Ca2ϩ ([Ca2ϩ]mt) and release a larger amount of Ca2ϩ from intracellular stores following stimulation of plasma membrane receptors coupled to IP3 generation [15]
Summary
The increased Ca2ϩ uptake is likely to depend on a higher efficiency of the SERCA pumps because the level of SERCA protein expression did not differ between P2X7- and mock transfected cells (Fig. 3C). Differences in [Ca2ϩ]mt levels depended on a functional P2X7 receptor as preincubation in the presence of oATP (2 h) equalized basal as well as carbacholstimulated [Ca2ϩ]mt of P2X7- and mock-transfected HEK293 cells (Fig. 1B). P2X7 Transfectants Are Resistant to Various Apoptotic Stimuli—We investigated whether HEK293-P2X7R cell clones were more resistant to cell death induced by differextent than by preincubation in the presence of oATP This is likely due to the brief (10 min) incubation under Ca2ϩ-free conditions. At variance with HEK293P2X7, P2X7 transfection did not increase basal [Ca2ϩ]mt, a difference likely caused by the fact that NIH3T3 cells were transiently transfected, while HEK293 cells were stably transfected
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