ERK inhibition increases ROS in alveolar macrophages. species, caspase activation was prevented, though necrotic pathways continued to induce cell death. This suggests that mitochondrial dysfunction caused by ERK inhibition generates both apoptotic and necrotic cell death-inducing pathways. As a composite, these data demonstrate a novel mitochondrial role for ERK in maintaining mitochondrial membrane potential and ATP production in human alveolar macrophages. to obtain cell pellets. The pellets were frozen at ?80 C. Pellets were thawed and homogenized in 50 mM potassium phosphate buffer, pH 7.8, containing 1.34 mM diethylenetriaminepentaacetic acid. Total glutathione content was determined by the method of Spitz (41). Reduced glutathione (GSH) and oxidized glutathione (GSSG) were distinguished by addition of 2 l of a 1:1 mixture of 2-vinylpyridine and ethanol per 50 l of sample followed by incubation for 1 h and assay as described previously by Griffith (42). All glutathione determinations were normalized to the protein content of whole homogenates using the method of Lowry (43). Transmission Electron Microscopy Samples were fixed overnight with 2.5% glutaraldehyde in 0.1 M cacodylate buffer. Post fixation was carried out for 1 hour at room temperature with a buffered 1% osmium tetroxide solution reduced with 1.5% potassium ferrocyanide. Samples were en bloc stained with 2.5% uranyl acetate. Cells were then rinsed and dehydrated using gradually increasing concentrations of acetone to 100%. Infiltration of Spurrs epoxy resin and acetone were carried out over several days to 100% resin and cured overnight in a 70C oven. Sections of 100nm thickness were cut using an Ultracut E ultramicrotome (Reichert-Jung). Grids were then counterstained with 5% uranyl acetate for 12 minutes and Reynolds lead citrate for 5 minutes. Samples were imaged using a Hitachi H-7000 transmission electron microscope. Phagocytosis Assay To evaluate bacterial phagocytosis by alveolar macrophages, cells were cultured in chamber slides (Lab Tek 4 chamber slides) for 2 hours with and without treatments and then exposed to GFP tagged e.coli. at a ratio of 25 bacteria per 1 cell. Cells and bacteria were incubated for a further 30 minutes. Non-phagocytosed cells were washed off by vigorously washing with PBS times 6. Images were obtained using an inverted fluorescent microscope (Zeiss) and then counts of bacteria per cell performed on random fields (fifty cells per group). In some cases, adherent but not phagocytosed bacteria were killed with gentamycin and then remaining bacteria quantified using bacterial plate counts. The data obtained from these studies was not different than those obtained using fluorescent analysis (data not shown). RESULTS Alveolar macrophages depend on mitochondria and the electron transport change for ATP production Studies extending back almost a century have suggested that both macrophages and neutrophils depend on cytosolic glycolysis for the generation of ATP (44-47). This includes macrophages found at sites of inflammation or wound repair that often depend on anaerobic glycolysis for ATP production (44, 45, 48). To determine the source of ATP in human alveolar macrophages, we cultured newly isolated alveolar macrophages with and without a number of inhibitors of mitochondrial ATP production. Oligomycin is an inhibitor of the ATP synthase subunit (F(1)F(0)) (49). Rotenone inhibits complex I of the electron transport chain (leading to generation Rabbit polyclonal to HRSP12 of reactive oxygen species (ROS)) (50, 51). CCCP is an uncoupler that disperses the proton gradient that drives ATP synthase without interfering directly with the ETC (52). Alveolar macrophages were treated with oligomycin (0.5 uM), rotenone (2.5.The data is presented as arbitrary fluorescent units. Both ERK inhibition and mitochondrial blockers induced loss of plasma membrane permeability and cell death. The cell death induced by ERK inhibition had hallmarks of both apoptotic (caspase activation) and necrotic (ATP loss) cell death. By blocking ERK-inhibition induced reactive oxygen species, caspase activation was prevented, though necrotic pathways continued to induce cell death. This suggests that mitochondrial dysfunction caused by ERK inhibition generates both apoptotic and necrotic cell death-inducing pathways. As a composite, these data demonstrate a novel mitochondrial role for ERK in maintaining mitochondrial membrane potential and ATP production in human alveolar macrophages. to obtain cell pellets. The pellets were frozen at ?80 C. Pellets were thawed and homogenized in 50 mM potassium phosphate buffer, pH 7.8, containing 1.34 mM diethylenetriaminepentaacetic acid. Total glutathione content was determined by the method of Spitz (41). Reduced glutathione (GSH) and oxidized glutathione (GSSG) were distinguished by addition of 2 l of a 1:1 mixture of 2-vinylpyridine and ethanol per 50 l of sample followed by incubation for 1 h and assay as described previously by Griffith (42). All glutathione determinations 48740 RP were normalized to the protein content of whole homogenates using the method of Lowry (43). Transmission Electron Microscopy Samples were fixed overnight with 2.5% glutaraldehyde in 0.1 M cacodylate buffer. Post fixation was 48740 RP carried out for 1 hour at room temperature with a buffered 1% osmium tetroxide solution reduced with 1.5% potassium ferrocyanide. Samples were en bloc stained with 2.5% uranyl acetate. Cells were then rinsed and dehydrated using gradually increasing concentrations of acetone to 100%. Infiltration of Spurrs epoxy resin and acetone were carried out over several days to 100% resin and cured overnight in a 70C oven. Sections of 100nm thickness were cut using an Ultracut E ultramicrotome (Reichert-Jung). Grids were then counterstained with 5% uranyl acetate for 12 minutes and Reynolds lead citrate for 5 minutes. Samples were imaged using a Hitachi H-7000 transmission electron microscope. Phagocytosis Assay To evaluate bacterial phagocytosis by alveolar macrophages, cells were cultured in chamber slides (Lab Tek 4 chamber slides) for 2 hours with and without treatments and then exposed to GFP tagged e.coli. at a ratio of 25 bacteria per 1 cell. Cells and bacteria were incubated for a further 30 minutes. Non-phagocytosed cells were washed off by vigorously washing with PBS times 6. Images were obtained using an inverted fluorescent microscope (Zeiss) and then counts of bacteria per cell performed on random fields (fifty cells per group). In some cases, adherent but not phagocytosed bacteria were killed with gentamycin and then remaining bacteria quantified using bacterial plate counts. The data obtained from these studies was not different than those obtained using fluorescent analysis (data not shown). RESULTS Alveolar macrophages depend on mitochondria and the electron transport change for ATP production Studies extending back almost a century have suggested that both macrophages and neutrophils depend on cytosolic glycolysis for the generation of ATP (44-47). This includes macrophages found at sites of inflammation or wound repair that often depend on anaerobic glycolysis for ATP production (44, 45, 48). To determine the source of ATP in human alveolar macrophages, we cultured newly isolated alveolar macrophages with and without a number of inhibitors of mitochondrial ATP production. Oligomycin is an inhibitor of the ATP synthase subunit (F(1)F(0)) (49). Rotenone inhibits complex I of the electron transport chain (leading to generation of reactive oxygen species (ROS)) (50, 51). CCCP 48740 RP is an uncoupler that disperses the proton gradient that drives ATP synthase without interfering directly with the ETC (52). Alveolar macrophages were treated 48740 RP with oligomycin (0.5 uM), rotenone (2.5 uM) and CCCP (10 uM). Combined ATP levels from both intracellular and extracellular sources were measured with a chemiluminescence reagent at various time points. ATP levels rapidly disappeared with all three exposures (Figure 1A). This data demonstrates that ATP levels in alveolar.