Regulation of phenobarbital induction of the cytochrome P450 2b9/10 genes in primary mouse hepatocyte culture - Involvement of calcium- and cAMP-dependent pathways

Phenobarbital (PB) has long been known as an inducer of drug-metabolizing e nzymes in liver, but the molecular mechanism underlying this induction is s till poorly understood. Using primary mouse hepatocyte culture, we have inv estigated the possible involvement of different regulatory pathways in PB a ction, by exposing PB-treated cells to various protein kinase/phosphatase m odulators. Our results showed a negative role of the cAMP-dependent pathway , as treatment with cAMP-dependent protein kinase (PKA) activators (10 mu M dibutyryl-cAMP and 50 mu M forskolin) dramatically inhibited PB-induced Cy p2b9/10 mRNA accumulation, whereas PKA inhibitor potentiated the PB respons iveness of this gene. The cGMP-dependent protein kinase (PKG) seems to play a positive role as PKG inhibitor reduced the PB-induced level of Cyp2b9/10 mRNA. We also obtained two lines of evidence for the involvement of Ca2+ i n modulating PB action. Firstly, measurements of intracellular Fura-2 fluor escence ratio in murine hepatocytes showed that long-term PB incubation (24 and 48 h) led to a significant increase of [Ca2+](i). Secondly, treatment with an intracellular Ca2+ chelator (BAPTA-AM) nearly completely abolished PB-induced Cyp2b9/10 expression. Ca2+ thus appeared to mediate PB action li kely via Ca2+/calmodulin-dependent protein kinase II, as KN62, a specific i nhibitor of this enzyme, also dramatically inhibited PB induction of the Cy p2b9/10 genes.