CRITICAL ROLE OF EXTRACELLULAR-MATRIX OIL INDUCTION BY PHENOBARBITAL OF CYTOCHROME-P450 2B1 2 IN PRIMARY CULTURES OF ADULT-RAT HEPATOCYTES/

BACKGROUND: Although it has been known for more than three decades tha t administration of lipophilic chemicals, including phenobarbital, pro duces liver hypertrophy, proliferation of smooth endoplasmic reticulum , and induction of liver microsomal enzymes such as cytochromes P450 ( CYP) 2B1 and 2B2, the mechanism of this adaptive response remains larg ely unknown. An important advance was the recognition that, unlike cul tures of continuously proliferating liver cell lines or cultures of pr imary non-proliferating adult rat hepatocytes maintained on either pla stic or collagen-coated dishes, hepatocytes cultured on a basement mem brane gel, Matrigel, formed rounded clusters and permitted phenobarbit al-mediated induction in vitro of CYP 2B1/2 mRNAs and immunoreactive p roteins (1). EXPERIMENTAL DESIGN AND RESULTS: We cultured adult rat he patocytes on Type I collagen (Vitrogen) and allowed the cells to sprea d, flatten, and firmly attach to the substratum. Subsequent incubation in medium containing Matrigel as a soluble component, fully restored, in a dose-dependent manner, the ability to respond to phenobarbital w ith induction of CYP 2B1/2 mRNAs. Repeating this experiment with mediu m containing equivalent amounts of purified laminin, a major component of Matrigel, or with YIGSR or SIKVAV, two peptides known to mimic var ious activities of laminin, similarly restored phenobarbital responsiv eness to hepatocytes cultured on Vitrogen. In contrast, use of equal a mounts of SHA-23, a scrambled peptide relevant to SIKVAV, produced no such effect. None of these treatments caused a rounding or any other o bservable change in the flattened, cellular morphology, making it unli kely that cell spreading or alterations in cell shape account for loss of such differentiated liver functions as phenobarbital induction of CYP 2B1/2 mRNAs in cultured hepatocytes on Vitrogen. Hepatocytes cultu red on Matrigel in the presence of either colchicine, cytochalasins B and D, nocodazole, or taxol did not show induction of 2B1/2 mRNAs by p henobarbital specifically, while the amounts of both albumin and gluco se-6-phosphate dehydrogenase (G6PD) mRNAs were unaffected. CONCLUSIONS : We conclude that the process by which phenobarbital induces 2B1/2 mR NAs in hepatocytes appears to require highly concerted effects of spec ific extracellular components prominently involving laminin. This like ly occurs through a signal transduction process requiring probably bot h microfilament and microtubular integrity.