ATP-evoked Ca2+ transients and currents in murine thymocytes: possible role for P2X receptors in death by neglect

Abstract

The P2X family of ATP receptors (P2XR) have been implicated in thymocyte death in vitro and in vivo. We characterized ATP-evoked Ca 2+ transients and membrane currents in thymocytes to better understand the role of P2XR during thymocyte development. ATP 4-, but not UTP or GTP, activated a sustained non-selective cation current in voltage-clamped CD4 -CD8 -and CD4 + CD8 + thymocytes that was reversed by apyrase, which hydrolyzes ATP, and by the P2XR antagonists suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). The more selective P2XR agonist § g -methylene ATP activated a smaller rapidly decaying current in both thymocyte populations. Reverse transcription-PCR results indicate that P2X1, P2X2, P2X6, and/or P2X7 are expressed in thymocytes. Finally, we used PPADS to examine the role of P2XR during thymocyte development in situ. PPADS-treated thymi yielded significantly more thymocytes (38 %), due to a selective increase in CD4 + CD8 + cells. Together these data suggest that one or more PPADS-sensitive P2XR (P2X1, P2X2, P2X7) are involved in thymocyte apoptosis, and we propose more specifically a role associated with death by neglect.

Extracellular ATP Induces Phosphatidylserine Externalization Earlier than Nuclear Apoptotic Events in Thymocytes

“…ATP ec (1 mM) triggered a Ca 2+ influx without Ca 2+ release from intracellular stores as previously reported5 (data not shown). Figure 2A shows that ATP ec (1 mM), in parallel to PS externalization, induced NBD‐PC internalization, consistent with the occurrence of a general phospholipid scrambling already described during apoptosis 2.…”

ORAI1 and STIM1 deficiency in human and mice: roles of store‐operated Ca2+ entry in the immune system and beyond

“…Details of how these receptors induce Ca 2+ signals and which Ca 2+ channels are activated is not well understood in many cases, although CRAC channel currents were observed in neutrophils in response to interleukin‐8 (IL‐8) treatment (11) and dendritic cells following passive store depletion (12). Other channels that may be involved in Ca 2+ influx in immune cells include TRP (transient receptor potential) channels (13, 14), the adenosine triphosphate (ATP)‐responsive P2Y and P2X purinoreceptors expressed on T cells and mast cells (15–18), and – although controversial – voltage‐gated Ca 2+ channels (19, 20). While TRP channels have not been shown to function as Ca 2+ ‐selective cation channels in T cells, it is of note that TRPC1 could be co‐immunoprecipitated together with STIM1 and ORAI1 in HEK293 and salivary gland cells (22) and that STIM1 was shown to gate TRPC1 and other TRPC channels (21).…”

Immunodeficiency due to defects in store‐operated calcium entry

“…1). Other Ca 2+ permeable channels and pathways have, however, been described as well such as P2X receptors in mast cells and T cells, [47][48][49][50] TRPC channels and voltage-gated Ca 2+ channels. [51][52][53] The contribution of these channels to immune responses in vivo remains largely unclear.…”