Role of heme in phenobarbital induction of cytochromes P450 and 5-aminolevulinate synthase in cultured rat hepatocytes maintained on an extracellular matrix.
When hepatocytes are cultured on matrigel, a reconstituted basement membrane matrix, mRNAs for cytochrome P450 class IIB1/2 and class III genes can be induced by treatment with phenobarbital. We took advantage of this new system to critically evaluate the role of heme as a regulator of these cytochromes P450 and of 5-aminolevulinate synthase (ALA-S), the rate-limiting enzyme in heme biosynthesis. Phenobarbital treatment of rat cultures increased the total amount of cytochrome P450, activities catalyzed by IIB1/2 (benzyloxy- and pentoxyresorufin O-dealkylases) and ALA-S activity, and ALA-S mRNA. Treatments with phenobarbital combined with succinyl acetone, an inhibitor of heme biosynthesis at the step of 5-aminolevulinate dehydrase, blocked the induction of the proteins for cytochrome P450IIB1/2 and cytochrome P450IIIAI, as indicated by spectral, immunological, and enzymatic assays. However, at the same time, succinyl acetone cotreatment failed to inhibit the induction of the mRNAs for cytochrome P450IIB1/2 and cytochrome P450IIIA. Lack of effect on the cytochrome P450 mRNAs was selective inasmuch as treatment with phenobarbital combined with succinyl acetone synergistically increased both ALA-S activity and ALA-S mRNA, presumably by blocking formation of heme, the feedback repressor of ALA-S. Indeed, the increase in ALA-S mRNA caused by the combined treatment was abolished by adding heme itself to the cultures. In contrast to earlier concepts, we conclude that in the intact hepatocyte, phenobarbital-induced cytochrome P450 induction is independent of changes in heme synthesis.
Sinclair
P R
PR
Veterans Administration, White River Junction, Vermont 05001.
Schuetz
E G
EG
Bement
W J
WJ
Haugen
S A
SA
Sinclair
J F
JF
May
B K
BK
Li
D
D
Guzelian
P S
PS
D000624:5-Aminolevulinate Synthetase / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D003470:Culture Media; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D004790:Enzyme Induction / Q000187:drug effects; D005109:Extracellular Matrix; D006418:Heme / Q000502:physiology*; D006537:Heptanoates / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D010634:Phenobarbital / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats; D011916:Rats, Inbred F344
D003470:Culture Media; D006537:Heptanoates; D012333:RNA, Messenger; D006418:Heme; C020804:succinylacetone; D003577:Cytochrome P-450 Enzyme System; D000624:5-Aminolevulinate Synthetase; D010634:Phenobarbital