Such activation of NFB by NO may result in a positive feedback system (Nam et al
Such activation of NFB by NO may result in a positive feedback system (Nam et al., 2004). astrocyte swelling. Western blot analysis of cultured astrocytes showed a significant increase in NFB nuclear translocation (a measure of NFB activation) from 12 h to 2 days after treatment with NH4Cl (5 mM). Cultures treated with antioxidants, including superoxide dismutase, catalase and vitamin E, as well as the MAPKs inhibitors SB239063 (an inhibitor of p38-MAPK), and SP600125 (an inhibitor of c-Jun N-terminal kinase, JNK) significantly diminished NFB activation by ammonia, assisting a role of oxidative stress and MAPKs in NFB activation. The activation of NFB was associated with improved iNOS protein expression and NO generation, and these changes were clogged by BAY 11-7082, an inhibitor of NFB. Additionally, ammonia-induced astrocyte swelling was inhibited from the NFB inhibitors BAY 11-7082 and SN-50, therefore implicating NFB in the mechanism of astrocyte swelling. Our studies show that cultured astrocytes exposed to ammonia display NFB activation, which is likely a consequence of oxidative stress and Mouse monoclonal to STAT3 activation of MAPKs. NFB activation appears to contribute to the mechanism of ammonia-induced astrocyte swelling, apparently through its upregulation of iNOS protein expression and the subsequent generation of nitric oxide. 1987; Swain 1991). Ammonia has been strongly implicated as a major toxin in liver failure (for review, Oxyclozanide see Albrecht and Jones, 1999; Hazell and Butterworth, 1999), and ammonia offers been shown to induce astrocyte swelling in models of hyperammonemia (Voorhies and were approved by the local animal care committee (IACUC). Western blots Samples of astrocytes were analyzed for iNOS and NFB protein content by Western blots as explained previously (Jayakumar (2005). In brief, astrocytes were washed twice with phosphate-buffered saline (PBS), then harvested in 1 ml PBS and Oxyclozanide centrifuged at 3,000 rpm for 3 min at 4C. The cell pellet was resuspended in 200 l chilly buffer A, consisting of 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 M dithiothreitol (DTT), and a complete protease inhibitor cocktail (Roche, Mannheim, Germany). The pellet was then incubated on snow for 15 min to allow cells to swell, after which 15 l of 10% NP-40 was added, and the sample was vortexed thoroughly for 40 sec. An aliquot of homogenate was utilized for protein measurement. Equal amount of protein was then centrifuged at 3,000 rpm for 3 min at 4C. The producing nuclear pellet was resuspended in 30 l chilly buffer B consisting of 20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 M DTT, and protease inhibitors. The pellet was then incubated on snow and vortexed for 15 sec every 2 min for up to 15 min. The nuclear draw out was then centrifuged at 13,000 rpm for 5 min at 4C. The supernatant (comprising the nuclear extract) was loaded on an SDS-polyacrylamide gel, and Western blot analysis with NFB antibody was performed as explained above. The quality of the nuclear draw out was analyzed by propidium iodide staining, which indicated a purity of 92C96%. Immunofluorescence of NFB Immunocytochemistry was performed on cultured astrocytes to directly visualize the translocation of the p65 subunit of NFB after ammonia treatment. Cultures were fixed with 4% paraformaldehyde for 30 min at RT. To prevent non-specific binding, cells were in the beginning treated with PBST (PBS + 0.1% Triton X-100) containing 10% normal goat serum for 30 min, and then incubated overnight having a rabbit polyclonal antibody against p65 (H-286) (cat# sc-7151). Following washing with PBST, astrocytes were incubated having a goat anti-rabbit fluorescent antibody (Alexa Fluor 488, Molecular Probes, Eugene, OR, USA) for 25 min. Cultures were also stained with propidium iodide (PI; 2 g/ml) to determine the total number of nuclei and the results were visualized having a Nikon Diaphot inverted fluorescent microscope equipped Oxyclozanide with multivariant fluorescent filters. Both cytoplasmic and nuclear NFkB staining was observed in ammonia-treated as well as in control cultures, but intensity of nuclear staining was improved in ammonia-treated cells. However, to more clearly ascertain variations in nuclear fluorescence between these two organizations, the intensity threshold value of nuclei was arranged as low as possible in the control cultures so as to avoid any cytoplasmic staining. The same intensity threshold was then applied to the ammonia-treated cultures. The number of NFkB-positive nuclei was counted from 5 random fields of the PI image (10x objective) using Sigma Scan Pro 5, and the results were indicated like a percent modify over control. The data were analyzed using a chi-square test. Cell volume measurement Astrocyte cell volume (intracellular water space) was.