The role of protein kinase C in the increased generation in isolated rat hepatocytes of the hydroxyl radical by puromycin aminonucleoside

Puromycin aminonucleoside (PAN) has been known to induce proteinuria. The i ncreased generation of reactive oxygen species (ROS) has been implicated in this toxicity of PAN. We have reported that PAN increases the synthesis of methylguanidine (MG) and creatol which are the products of the reaction of creatinine and the hydroxyl radical in isolated rat hepatocytes. However, the mechanism for the increased ROS induced by PAN is still unclear. In thi s paper, we investigate the role of protein kinase C (PKC) on the PAN induc ed reactive oxygen generation in isolated rat hepatocytes. Isolated hepatoc ytes were incubated in Krebs-Henseleit bicarbonate buffer containing 3% BSA , 16.6 mM creatinine and tested reagents. MG and creatol were determined by high-performance liquid chromatography using 9,10-phenanthrenequinone for the post-labeling. PAN increased MG and creatol synthesis in isolated rat h epatocytes by 60%. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochl oride (H-7), a PKC inhibitor, at 10 and 100 mu M significantly inhibited MG and creatol synthesis with or without PAN. The inhibition rate is dose dep endent from 10 to 100 mu M. H1004, a reagent used as control for H-7, did n ot affect (at 10 mu M) or increased little (at 100 mu M) the synthesis of M G and creatol. Ro31-8425, a potent PKC inhibitor, significantly inhibited ( at 10 mu M) MG synthesis in the presence of PAN. PKC in the membrane fracti on, a marker of PKC activation, increased over the initial concentration by a factor of 1.65-fold at 60 min incubation and 2.16-fold at 120 min with P AN, while it changed little without PAN. These results indicate that PAN ac tivates PKC resulting in increased hydroxyl radical generation in isolated rat hepatocytes.