CHARACTERIZATION OF A PRETRANSCRIPTIONAL PATHWAY FOR INDUCTION BY PHENOBARBITAL OF CYTOCHROME-P450 3A23 IN PRIMARY CULTURES OF ADULT-RAT HEPATOCYTES

Our laboratory has proposed that phenobarbital (PB), a typical lipophi lic agent that induces some members of the supergene family of liver m icrosomal cytochromes P450 (e.g., CYP2B1/2 and CYP3A23), acts through a complex process inhibitable by the presence of growth hormone (GH), the absence of some components of the extracellular matrix, or a disru pted cytoskeleton. To verify that these manipulations of the culture e nvironment block specific steps in the PB induction pathway rather tha n simply exerting nonspecific or toxic effects on CYP2B1/2 gene transc ription, we have now examined PE induction of CYP3A23, a gene known to also be transcriptionally activated by dexamethasone (DEX) through a ''nonclassical'' pathway apparently involving the glucocorticoid recep tor. We found that in primary cultures of adult rat hepatocytes treate d with PB, induction of CYP3A23 mRNA, just as we reported for inductio n of CYP2B1/2 mRNA, required the use of Matrigel (a reconstituted base ment membrane) and was blocked by the presence of cytoskeletal inhibit ors (colchicine or cytochalasins) or of physiologic concentrations of GH in the culture medium. Moreover, PB induction of CYP3A23 and of CYP 2B1/2 mRNAs was greatly diminished by inhibitors of cAMP-dependent pro tein kinase (PKA). In striking contrast, induction of CYP3A23 mRNA by DEX was unaffected by any of these alterations of the culture conditio ns that block its induction by PB. We conclude that the effects of ext racellular matrix, GH, disruption of the cytoskeleton, and activation of cAMP-dependent protein kinase, pharmacologically define multiple, p retranscriptional steps in the pathway(s) for PB induction of liver cy tochromes P450. (C) 1997 Academic Press.