Abstract

The N-demethylation of N,N'-dimethylphenobarbital by isolated rat hepatocytes was increased severalfold by pretreatment of animals with sodium phenobarbital (75 mg/kg/day ip for 3 days). The apparent extent of induction depended on the length of the cell incubation, being 3-fold when calculated after a 5-min incubation and 5-fold when calculated after a 60-min incubation. This difference was due to the fact that metabolism by "induced" hepatocytes more closely approached linearity over a 60-min period. Microsomal cytochrome P-450 levels were increased 3-fold upon phenobarbital treatment; during incubations of less than 30 min duration, increased N-demethylase activity could be entirely accounted for by this increase in cytochrome P-450. When protein levels were measured directly in microsomes isolated from hepatocyte homogenates, the levels in "induced" hepatocytes were approximately 45% higher than control. However, phenobarbital treatment had no significant effects on total cellular protein or on the protein distribution in various subcellular fractions when appropriate adjustments were made for apparent microsomal sedimentation with the 9000 g pellet. These data suggest that hepatic microsomes prepared from "induced" hepatocytes may sediment differently from those from untreated animals. The N-demethylation of N,N'-dimethylphenobarbital is an excellent model reaction to study in isolated hepatocytes because the primary metabolite, N-methylphenobarbital, is not further metabolized to an appreciable extent.