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Aim. To investigate the role of Ca2+ release-activated Ca2+ (CRAC) channels in the ROS production in macrophages. Methods. The intracellular [Ca2+]i was analyzed by confocal laser microscopy. The production of ROS was assayed by flow cytometry. Results. Both LPS and thapsigargin induced an increase in intracellular [Ca2+]i, either in the presence or absence of extracellular Ca2+ in murine macrophages. The Ca2+ signal was sustained in the presence of external Ca2+ and only initiated a mild and transient rise in the absence of external Ca2+. CRAC channel inhibitor 2-APB completely suppressed the Ca2+ entry signal evoked by thapsigargin, and suppressed approximately 93% of the Ca2+ entry signal evoked by LPS. The increase in intracellular [Ca2+]i, was associated with increased ROS production, which was completely abolished in the absence of extracellular Ca2+ or in the presence of CRAC channel inhibitors 2-APB and Gd3+. The mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy- phenylhydrazone and the inhibitor of the electron transport chain, antimycin, evoked a marked increase in ROS production and completely inhibited thapsigargin and LPS-evoked responses. Conclusions. These findings indicate that the LPS-induced intracellular [Ca2+]i increase depends on the Ca2+ entry through CRAC channels, and close functional coupling between CRAC and ROS production in murine macrophages. [ABSTRACT FROM AUTHOR]

Aim. To investigate the role of Ca2+ release-activated Ca2+ (CRAC) channels in the ROS production in macrophages. Methods. The intracellular [Ca2+]i was analyzed by confocal laser microscopy. The production of ROS was assayed by flow cytometry. Results. Both LPS and thapsigargin induced an increase in intracellular [Ca2+]i, either in the presence or absence of extracellular Ca2+ in murine macrophages. The Ca2+ signal was sustained in the presence of external Ca2+ and only initiated a mild and transient rise in the absence of external Ca2+. CRAC channel inhibitor 2-APB completely suppressed the Ca2+ entry signal evoked by thapsigargin, and suppressed approximately 93% of the Ca2+ entry signal evoked by LPS. The increase in intracellular [Ca2+]i was associated with increased ROS production, which was completely abolished in the absence of extracellular Ca2+ or in the presence of CRAC channel inhibitors 2-APB and Gd3+. The mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhydrazone and the inhibitor of the electron transport chain, antimycin, evoked a marked increase in ROS production and completely inhibited thapsigargin and LPS-evoked responses. Conclusions. These findings indicate that the LPS-induced intracellular [Ca2+]i increase depends on the Ca2+ entry through CRAC channels, and close functional coupling between CRAC and ROS production in murine macrophages. [ABSTRACT FROM AUTHOR]