Inflammasome Activation Purinergic Receptors Involved in ATP-induced ROS Generation It has been proposed that extracellular ATP induces ROS MGCD516 web production through ligation of the ATP-gated P2X7 ion channel, in association with the pore-forming hemi-channel, pannexin-1, in macrophages and neurons. To investigate whether ATP-induced ROS production in GEC takes place through P2X7, we examined the gene expression levels of purinergic receptors in HIGK cells. In agreement with our previous description of primary GEC, the HIGK cells express P2X1 throuh P2X7. In addition, the HIGK cells express pannexin-1. P2X- and pannexin-1-dependent responses in HIGK cells were next examined by fluorescence microscopy. Consistent with previous results, 3 mM ATP stimulated a large level of ROS production, suggesting that ATP mediates ROS production through P2X7 ligation. A role for P2X7 was further confirmed by showing that ATP-induced ROS 19053768 production was inhibited by pretreatment with the P2X7 and pannexin-1 antagonists, oxATP and probenecid, respectively. Moreover, as illustrated in activation in GEC takes place through P2X4/P2X7 ligation. Using ELISA to measure secretion of activated caspase-1, we observed that treatment of GEC with 100 mM ATP was insufficient for caspase-1 activation, even though ROS generation was induced. In contrast, 3 mM ATP treatment resulted in high levels of caspase-1 activation in GEC stably-expressing the control shRNA; but the activation of caspase-1 by 3 mM ATP treatment was abrogated when either P2X4 or P2X7 were depleted in GEC. Thus, treatment with 3 mM ATP induced ROS production via the P2X4/P2X7 complex and activated the NLRP3 inflammasome. However, 100 mM ATP stimulation induced ROS generation through P2X4 ligation, but stimulation with this concentration of ATP was not sufficient to activate the inflammasome. The non-redundant roles of P2X4 and P2X7 in ATP-induced ROS generation led us to hypothesize that P2X4 and P2X7 may be associated in the membrane and function as a physical complex in ATP-mediated responses in GEC. Therefore, we examined physical associations between P2X4 and P2X7 in GEC by performing co-immunoprecipitation experiments. After precipitating endogenous P2X4 using an anti-P2X4 antibody, we observed 17372040 that P2X7 and pannexin-1 were detected in the immunoprecipitate. Taken together, these data indicate that P2X4, P2X7, and pannexin-1 form a heterocomplex in GEC, and play non-redundant roles in ATP-induced ROS generation. ATP Ligation of P2X4/P2X7/Pannexin-1 Contributes to Secretion of pro-inflammatory Cytokines Secretion in Primary GEC Infected with P. gingivalis Confirmation by RNA Interference for Role of P2X7, P2X4 and Pannexin-1 in ATP-Mediated ROS Production As inhibitor studies suggested that P2X4 may be involved in ATP-dependent ROS responses, we examined this unexpected result by stably depleting P2X4, P2X7 and pannexin-1 by lentiviral delivery of specific shRNA. Specific cepletion efficiency in each cell line was validated individually by qPCR, as we have previously done to show specific depletion of purinergic receptors by RNA interference. As shown in ATP Ligation by P2X4/P2X7/Pannexin-1 Complex Leads to Inflammasome Activation in GEC We previously showed that ATP treatment of GEC leads to NLRP3 inflammasome activation. As ROS production has been associated with inflammasome and caspase-1 activation, we evaluated whether ATP-mediated caspase-1 P2X4 Modulates ATP-Induced Inflammasome Activation P2X7

The same panel shows EAAC1 immunoreactivity in different rat tissues

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