Modulation of murine phenobarbital-inducible CYP2A5, CYP2B10 and CYP1A enzymes by inhibitors of protein kinases and phosphatases.

Phenobarbital causes a multitude of effects in hepatocytes, including increased cell proliferation, inhibition of apoptosis and upregulation of xenobiotic and endobiotic metabolizing enzymes. In this study, the involvement of several protein kinase and phosphatase pathways on constitutive and phenobarbital-induced activities of CYP2A5, CYP2B10 and CYP1A1/2 in primary mouse hepatocytes was determined using well-defined chemical modulators of intracellular protein phosphorylation and desphosphorylation events. A 48-h treatment of the hepatocytes with 2-aminopurine, a nonspecific serine/threonine kinase inhibitor, elicited dose-dependent increases in both basal and phenobarbital-induced CYP2A5 catalytic activity (assayed as coumarin 7-hydroxylation), the maximal induction being 60-fold greater than the control value upon cotreatment with 1.5 mM phenobarbital and 10 mM 2-aminopurine. In contrast, phenobarbital induction of CYP2B10 (pentoxyresorufin O-deethylase) and CYP1A1/2 (ethoxyresorufin O-deethylase) activities were blocked by 2-aminopurine. Increases in CYP2A5 activity were also observed after exposure of the hepatocytes to other protein kinase inhibitors affecting the cell cycle, i.e. roscovitine, K-252a and rapamycin. Inhibitors of protein kinases A and C, as well as tyrosine kinases, did not appreciably affect CYP2A5 activity levels. The serine/threonine phosphatase inhibitors tautomycin, calyculin A and okadaic acid all reduced both basal and phenobarbital-induced CYP2A5, CYP2B10 and CYP1A1/2 activities. These results further strengthen the concept that hepatic CYP2A5 is regulated in a unique way compared with CYP2B10 and CYP1A.