Abstract
Extracellular ATP and other nucleotides act through specific cell surface receptors and regulate a wide variety of cellular responses in many cell types and tissues. In this study, we demonstrate that murine mast cells express several P2Y and P2X receptor subtypes including P2X(7), and describe functional responses of these cells to extracellular ATP. Stimulation of bone marrow-derived mast cells (BMMC), as well as MC/9 and P815 mast cell lines with millimolar concentrations of ATP, resulted in Ca(2+) influx across the cellular membrane and cell permeabilization. Moreover, brief exposures to ATP were sufficient to induce apoptosis in BMMCs, MC/9, and P815 cells which involved activation of caspase-3 and -8. However, in the time period between commitment to apoptosis and actual cell death, ATP triggered rapid but transient phosphorylation of multiple signaling molecules in BMMCs and MC/9 cells, including ERK, Jak2, and STAT6. In addition, ATP stimulation enhanced the expression of several proinflammatory cytokines, such as IL-4, IL-6, IL-13, and TNF-alpha. The effects of ATP were mimicked by submillimolar concentrations of 3-O-(4'-benzoyl)-benzoyl-benzoyl-ATP, and were inhibited by pretreatment of mast cells with a selective blocker of human and mouse P2X(7) receptor, 1[N,O-bis(5-isoquinolinesulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine, as well as oxidized ATP. The nucleotide selectivity and pharmacological profile data support the role for P2X(7) receptor as the mediator of the ATP-induced responses. Given the importance of mast cells in diverse pathological conditions, the ability of extracellular ATP to induce the P2X(7)-mediated apoptosis in these cells may facilitate the development of new strategies to modulate mast cell activities.
Highlights
The present work demonstrates that murine mast cells express a functional P2X7 receptor capable of initiating apoptosis after stimulation with millimolar concentrations of extracellular ATP
Notwithstanding the development of apoptosis, an up-regulation in the expression level of several proinflammatory cytokines was detected by RT-PCR and ELISA in bone marrowderived mast cells (BMMC) and MC/9 cells, including IL-4, IL-6, IL-13, and TNF-␣
The identification of P2X7 as a receptor subtype responsible for the observed effects of ATP was supported by several lines of experimental evidence: 1) only millimolar concentrations of ATP were effective in inducing apoptosis, whereas BzATP mimicked ATP action at submillimolar doses; 2) the treatment with ATP or BzATP triggered a rapid transmembrane Ca2ϩ influx through channels permeable to small cations and opened a nonselective pore capable of allowing an uptake of low molecular mass hydrophilic solutes; 3) the effects of ATP or BzATP on mast cells were abrogated by pretreatment with KN-62 or oxidized ATP (oATP); and 4) RT-PCR and Western blotting analysis confirmed the presence of P2X7 mRNA and protein in mast cells
Summary
BMMC, bone marrow-derived mast cell; oATP, oxidized ATP; KN-62, 1[N,O-bis(5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl]-4phenylpiperazine; BzATP, 3-O-(4Ј-benzoyl)-benzoyl-benzoyl-ATP; pTyr, phosphotyrosine; PARP, poly(ADP-ribose) polymerase; PI, propidium iodide; AFC, 7-amido4(trifluoromethyl)coumarin; CaMKII, Ca2ϩ/calmodulin-dependent protein kinase II. P2X7 receptor requires millimolar levels of ATP in the presence of divalent cations to achieve activation [19, 20], which leads to the formation of a nonselective cationic channel with low affinity for ATP and increased permeability to Ca2ϩ, intracellular depolarization, and equilibration of sodium and potassium gradients [21, 22]. The ability of extracellular ATP to induce apoptosis in BMMCs as well as in MC/9 and P815 mast cell lines through P2X7 receptor has not been investigated yet. We provide experimental evidence that extracellular ATP in millimolar range induces the P2X7-mediated apoptosis in BMMCs and MC/9 mast cells. In the time lag between the commitment to apoptosis and actual cell death, extracellular ATP stimulates the phosphorylation of ERK1/2, Jak, and STAT6 in mast cells, and transitorily up-regulates expression of several proinflammatory cytokines, such as IL-4, IL-6, IL-13, and TNF-␣
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