FIGURE 3.
Uncoupled eNOS contributes to Ang II-induced intracellular superoxide generation in MCs. A, eNOS is expressed in glomerular MCs. Left panel, immunoblot detection using anti-eNOS antibodies shows a 130–145-kDa band corresponding to the predicted molecular weight of the enzyme in glomerular endothelial cells and MCs. Right panel, immunofluorescence confocal microscopy using eNOS antibody and FITC-linked secondary antibodies showing eNOS distribution in MCs. B, MCs were untransfected (−) and transfected with control Scr (100 nm) or with sieNOS (100 nm). eNOS protein expression was determined by Western blot analysis. Transfection of MCs with sieNOS but not Scr reduces the 130–145-kDa band. C and D, MCs were untransfected or transfected with Scr or sieNOS and exposed to 1 μm Ang II for 10 min, 1 h, or 24 h. Intracellular superoxide generation was evaluated using DHE (10 μm, 30 min) and confocal microscopy (C) or DHE and a multiwell fluorescence plate reader (D) as described under “Experimental Procedures.” In C, the right panels represent a semiquantification of relative DHE fluorescence (arbitrary units). The values are the means ± S.E. from three independent experiments. **, p < 0.01 versus control. ##, p < 0.01 versus Ang II in untransfected cells. E and F, Ang II decrease the eNOS dimer to monomer ratio, a reflection of the disruption of eNOS dimer stability and eNOS uncoupling. Serum-deprived MCs were exposed to 1 μm Ang II for the indicated times, and samples were subjected to low temperature SDS-PAGE. The Western blots shown are representative of at least three independent experiments. The bottom panels represent the ratio of the intensity of eNOS dimer bands quantified by densitometry factored by the densitometric measurement of eNOS monomer band. The values are the means ± S.E. from three independent experiments. **, p < 0.01 versus control. G, treatment of the cells with Ang II (1 μm) for short or prolonged time periods did not increase the expression of iNOS or nNOS protein. Serum-deprived MCs were exposed to 1 μm Ang II for a short or prolonged time period. iNOS (top panel) or nNOS (bottom panel) proteins were determined by direct immunoblotting. Mθ, LPS-activated macrophage lysate used as positive control for iNOS. Rat brain was used as a positive control for nNOS.