Ma. Robin et al., Vesicular transport of newly synthesized cytochromes P4501A to the outsideof rat hepatocyte plasma membranes, J PHARM EXP, 294(3), 2000, pp. 1063-1069
Citations number
31
Categorie Soggetti
Pharmacology & Toxicology
Journal title
JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Anti-cytochrome P450 (CYP)1A2 autoantibodies are found in dihydralazine-ind
uced hepatitis, and CYPs2B and 2C have been shown to follow vesicular flow
to the plasma membrane (PM). However, it is unknown whether other CYPs foll
ow this route, whether NADPH-CYP reductase is present on the hepatocyte sur
face, and whether autoimmune hepatitis-inducing drugs increase PM CYPs. In
this study, we determined the transmembrane topology and transport of CYPs1
A in rat hepatocytes. In cultured hepatocytes, colchicine and other vesicul
ar transport inhibitors decreased PM CYPs1A assessed by flow cytometry. Col
chicine administration also decreased PM CYPs1A in vivo. Pulse chase experi
ments with [S-35]methionine showed that only the newly synthesized CYP mole
cules are transferred to the PM, whereas microsomal CYP1A2 was stably radio
labeled for several hours. In contrast, radiolabeled CYP1A2 reached the PM
and disappeared from the PM with half-lives of less than 30 min. Confocal m
icroscopy, 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 degrees C, before five washes and PM preparati
on, abolished PM CYPs1A-supported monooxygenase activity, indicating that P
M CYPs are mostly located on the external surface. Dihydralazine and other
CYPs1A inducers increased PM CYPs1A. In conclusion, newly synthesized CYPs1
A follow vesicular flow to the outside of the PM, and NADPH-CYP reductase a
lso is located on the hepatocyte surface. Dihydralazine administration incr
eases PM CYP1A2, its autoimmune target.