Involvement of P2X₄, P2X₇, and pannexin-1 in ATP-mediated ROS generation.

<p>(<b>A</b>) Total RNA from immortalized GEC was extracted and reversed transcribed (RT) to cDNA. The cDNA was used to perform PCR with the primers specific for the indicated genes, and the PCR products were finally visualized by EtBr staining. (<b>B</b>) GEC were treated with 100 µM or 3 mM ATP as indicated for 1 hour, and ROS production was measured with DCF staining and visualized by fluorescence microcopy. Quantification of the fluorescence in three independent experiments with SD is shown in (<b>C</b>). (D–E) ROS production was measured by DCF staining in GEC stimulated under different conditions. GEC were left untreated or stimulated with 20 µM nigericin or 3 mM ATP for 1 hour followed by fluorescence microscopy. Diminished ROS generation by different receptor antagonists was examined by pretreating cells with 100 µM oxATP for 30 minutes, 1 mM probenecid for 10 minutes, or 50 µM 5-BDBD or 100 µM PPADS for 15 minutes, followed by 3 mM ATP stimulation for 1 hour.