Hepatocyte cytoskeleton during ischemia and reperfusion - influence of ANP-mediated p38 MAPK activation

Figure 4 Analysis of the influence of ANP preconditioning on localization of F-actin in the liver by confocal microscopy. Livers were left untreated (A): preconditioned with 200 nmol/L ANP (B): 2 μmol/L SB203580 (C): or perfused with a combination of ANP (200 nmol/L) and SB203580 (2 μmol/L) (D): After ischemia (24 h, 4 °C), livers were snap-frozen, cut into 6-μm slices and stained with rhodamine-conjugated phalloidin (red, F-actin) and Hoechst 33342 (blue, nuclei) as described in “Materials and methods”. Liver sections were observed by confocal laser microscopy (63-fold amplification). Immunohistological staining of KCs in untreated (Co) and KC-depleted livers (E): rats received an intravenous injection of 900 µL NaCl (Co) or 900 µL liposomes (12 mmol/L Cl₂MBP) into the tail vein. After 48 h rat livers were perfused, snap-frozen and liver slices were stained with an antibody against the KC-specific surface marker ED2 (see “Methods”) to verify the Cl₂MBP-dependent depletion of the KCs. Dark staining represents KC (hallmarked with arrows). (F): KCs were depleted with Cl₂MBP as described under “Methods”. After 48 h control organs (Co) or KC-depleted livers (12 mmol/L Cl₂MBP) were perfused±ANP (200 nmol/L) for 20 min. Then p38 MAPK activity was investigated by in vitro phosphorylation assay (see “Methods”). Determination of density light units was performed by phosphorimaging and values of ANP-pretreated cells were divided by mean values of the respective control group. Columns show mean±SE of 4-5 independent perfusion experiments with ^bP<0.01 being statistically different from the respective control group.