Role of heme in phenobarbital induction of cytochromes P450 and 5-aminolevulinate synthase in cultured rat hepatocytes maintained on an extracellular matrix

When hepatocytes are cultured on matrigel, a reconstituted basement membrane matrix, mRNAs for cytochrome P450 class IIB1/2 and class III genes can be induced by treatment with phenobarbital. We took advantage of this new system to critically evaluate the role of heme as a regulator of these cytochromes P450 and of 5-aminolevulinate synthase (ALA-S), the rate-limiting enzyme in heme biosynthesis. Phenobarbital treatment of rat cultures increased the total amount of cytochrome P450, activities catalyzed by IIB1/2 (benzyloxy- and pentoxyresorufin O-dealkylases) and ALA-S activity, and ALA-S mRNA. Treatments with phenobarbital combined with succinyl acetone, an inhibitor of heme biosynthesis at the step of 5-aminolevulinate dehydrase, blocked the induction of the proteins for cytochrome P450IIB1/2 and cytochrome P450IIIAI, as indicated by spectral, immunological, and enzymatic assays. However, at the same time, succinyl acetone cotreatment failed to inhibit the induction of the mRNAs for cytochrome P450IIB1/2 and cytochrome P450IIIA. Lack of effect on the cytochrome P450 mRNAs was selective inasmuch as treatment with phenobarbital combined with succinyl acetone synergistically increased both ALA-S activity and ALA-S mRNA, presumably by blocking formation of heme, the feedback repressor of ALA-S. Indeed, the increase in ALA-S mRNA caused by the combined treatment was abolished by adding heme itself to the cultures. In contrast to earlier concepts, we conclude that in the intact hepatocyte, phenobarbital-induced cytochrome P450 induction is independent of changes in heme synthesis.