Dominique Pessayre Inserm Logo

Abstract

Anti-cytochrome P450 (CYP)1A2 autoantibodies are found in dihydralazine-induced hepatitis, and CYPs2B and 2C have been shown to follow vesicular flow to the plasma membrane (PM). However, it is unknown whether other CYPs follow this route, whether NADPH-CYP reductase is present on the hepatocyte surface, and whether autoimmune hepatitis-inducing drugs increase PM CYPs. In this study, we determined the transmembrane topology and transport of CYPs1A in rat hepatocytes. In cultured hepatocytes, colchicine and other vesicular transport inhibitors decreased PM CYPs1A assessed by flow cytometry. Colchicine administration also decreased PM CYPs1A in vivo. Pulse chase experiments with [35S]methionine showed that only the newly synthesized CYP molecules are transferred to the PM, whereas microsomal CYP1A2 was stably radiolabeled for several hours. In contrast, radiolabeled CYP1A2 reached the PM and disappeared from the PM with half-lives of less than 30 min. Confocal microscopy, biotinylation, and coimmunoprecipitation experiments showed that PM CYPs1A and CYP reductase are present on the cell surface, and that the reductase is closely associated with PM CYPs. Exposure of whole cells to an anti-CYP1A1/2 antibody at 4°C, before five washes and PM preparation, abolished PM CYPs1A-supported monooxygenase activity, indicating that PM CYPs are mostly located on the external surface. Dihydralazine and other CYPs1A inducers increased PM CYPs1A. In conclusion, newly synthesized CYPs1A follow vesicular flow to the outside of the PM, and NADPH-CYP reductase also is located on the hepatocyte surface. Dihydralazine administration increases PM CYP1A2, its autoimmune target.

Footnotes

  • Send reprint requests to: Dr. Dominique Pessayre, INSERM U481, Hôpital Beaujon, 100 boulevard du Général Leclerc, 92118 Clichy, France. E-mail:pessayre{at}bichat.inserm.fr

  • ↵1 This study was supported in part by the Bioavenir Institut National de la Santé et de la Recherche Médicale /Rhone Poulenc Rorer program (contract no. 95133) and the European Union BIOMED 2 program (contract BMH4-CT96- 0658).

  • Abbreviations:
    CYP
    cytochrome P450
    PM
    plasma membrane
    ER
    endoplasmic reticulum
    CPR
    NADPH-CYP reductase
    FITC
    fluorescein isothiocyanate
    EROD
    ethoxyresorufin O-deethylase
    • Received January 24, 2000.
    • Accepted May 25, 2000.
  • The American Society for Pharmacology and Experimental Therapeutics

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