2007a). m-Tyramine of TLR4 prevented the activation of cPLA2 and COX-2 as well as diminished PGE2 production, suggesting that interactive phosphorylation of TLR4CSrc regulated the pro-inflammatory response in astrocytes. Experiments with small interfering RNA knockdown of TLR4 in human astrocytes confirmed that silencing expression also abolished the interactive phosphorylation of both TLR4 and Src in the presence of ethanol. antagonist LPS were purchased from Sigma Aldrich (St. Louis, MO, USA). Cytotoxicity assay The fluorescence-based live/dead assay (Invitrogen Corporation, Carlsbad, CA, USA) determined astrocyte viability as per m-Tyramine manufacturers instructions. Primary astrocytes cultured on poly-D-lysine-coated 96-well plates (20,000 cells/well) were treated with concentrations of EtOH ranging from 10 to 300 mM for 48 h in culture. Following two washes with phosphate-buffered saline (PBS), cells were incubated with 2 M calcein AM and 4 M of ethidium homodimer (EthD-1) for 20 min at room temperature. Enzymatic conversion of the cell-permeable calcein AM to the fluorescent calcein determined the live cells. Cell death was identified by increased fluorescence resulting from the entry of EthD-1 across damaged cell membranes and binding to nucleic acids. Using a fluorescence plate reader (Molecular Devices, Sunnyvale, CA, USA), fluorescent calcein was detected at 490 nm excitation and 515 nm emission, while fluorescent EthD-1 was detected at 528 nm excitation and 617 nm emission. Results were expressed as percent of live cells (data not shown). ROS detection and CYP2E1 activity Primary astrocytes cultured in GNASXL 96-well plates (20,000 cells/well) were used to determine the changes in ROS levels detected by dichlorofluorescein-diacetate (DCF-DA) assay following the previously published method (Haorah et al. 2007a). The 105,000pellets containing astrocytic microsomal protein was used to assay CYP2E1 activity by hydroxylation of shows the Western blot analyses of TLR4 protein suppression by TLR4-specific siRNA transfection without altering the level of actin protein. Co-localization of TLR4 protein (nonsilencing control siRNA, EtOH + nonsilencing control siRNA, TLR4-specific siRNA transfection control, and EtOH + TLR4-specific siRNA transfection. b shows the Western blot analyses of p-Src Tyr416 protein suppression by TLR4-specific siRNA transfection without changing actin level. Co-localization of p-Src Tyr416 protein (nonsilencing control siRNA, EtOH + nonsilencing control siRNA, TLR4-specific siRNA transfection control, and EtOH + TLR4-specific siRNA transfection. Phosphorylated Src kinase ( em p-Src /em ) was probed by Src antibody specific to anti-phospho-Tyr416 (original magnification 20) Open in a separate window Fig. 9 Alcohol-induced TLR4 protein recruitment mediates the activation of Src kinase signaling pathway Discussion Alcohol abuse causes significant structural and functional alterations in the CNS (Harper et al. 2003); however, the underlying mechanisms of such effects are still largely unknown. We tested the idea that alcohol could increase the production of reactive metabolites (ROS, Ach) due to EtOH metabolism by CYP2E1 in astrocytes. These reactive metabolites could then activate (phosphorylate) Src through TLR4 recruitment, leading to the induction of PLA2 and COX activity and production m-Tyramine of pro-inflammatory PGE2. Pathophysiologically relevant concentration of 20 mM EtOH increased CYP2E1 activity paralleling enhanced ROS production (Fig. 1a, b) similar to the findings in rat astrocytes and neurons (Montoliu et al. 1995; Kapoor et al. 2006), suggesting that CYP2E1 indeed has a prominent role in ROS generation in human astrocytes. Our findings suggested that activation of NOX appeared to be the main source of ROS production because APC (NOX inhibitor) prevented the EtOH/Ach-induced increase in ROS level (Fig. 1b). We hypothesized that reactive EtOH metabolites could activate Src via TLR recruitment with subsequent activation of PLA2 and COX, leading to secretion of inflammatory PGE2. Indeed, treatment of astrocytes with the inhibitor, PP2 or AACOCF3, significantly reduced (71C73%) the EtOH/Ach-induced up-regulation of COX-2 protein level and subsequent PGE2 production. This was likely due to enhanced production of AA resulting from hydrolysis of phospholipids by PLA2. Subsequent metabolism of AA by COX-1 and -2 yielded PGE2 in the extracellular medium. Similar to our findings, Luo et al. (2001) showed that relatively high EtOH dose (50C100 mM) or even a physiologically relevant lower EtOH dose (20 mM, 0.1% em v /em / em w /em ) enhanced PGE2 production in astrocytes due to PLA2-mediated COX-2 activation (Luo et al. 2001). A most recent report by Lin et al. (2010) demonstrated similar.