FIGURE 4. Sub-cytotoxic 4-HNE treatment of primary rat hepatocyte cultures results in a concentration-dependent decrease in ERK-1/2 phosphorylation and activity that is inversely correlated with the increase in 4-HNE-ERK-1/2 adduct concentration.

The cytotoxicity of 4-HNE after 4 h of treatment on primary rat hepatocytes was determined over a range of physiologic concentrations. A, cytotoxicity shown as the ratio of release LDH activity over total cellular LDH activity, indicating a lack of cytotoxicity from 0 to 100 μm 4-HNE. These sub-cytotoxic concentrations were used for subsequent primary culture experiments. B, immunoblot indicating a concentration-dependent decrease in ERK-1/2 phosphorylation when compared with constitutive control levels (lane 1, 0 μm 4-HNE) (B-actin reprobe of stripped blots confirms equal loading). C, immunoblot analysis of total ERK-1/2 protein indicating no change with increasing concentrations of 4-HNE treatment (B-actin reprobe of stripped blots indicates equal loading). D, ERK-1/2 activity assay demonstrating the concentration-dependent suppression of immunoprecipitated ERK-1/2 collected from primary cultures exposed to increasing levels of 4-HNE to phosphorylate the ELK-1 substrate. D, inset, active, phosphorylated IP-ERK-1/2 from control hepatocytes is not affected by increasing concentrations of 4-HNE, indicating 4-HNE-mediated effects on ERK-1/2 result from interactions with the inactive, unphosphorylated ERK monomers. E, Western blot analysis of activity assay components probed for 4-HNE-protein adducts indicating an inverse correlation between ERK-1/2 phosphorylation and activity and the concentration-dependent increase in 4-HNE-adducted ERK-1/2 levels in primary hepatocytes. (*, p < 0.05, n = 9.)