Expression of 5-aminolaevulinate synthase and cytochrome P-450 mRNAs in chicken embryo hepatocytes in vivo and in culture. Effect of porphyrinogenic drugs and haem.
To examine current models for the co-ordinate regulation of 5-aminolaevulinate (ALA) synthase and cytochrome P-450 we have determined the effect of drugs, inhibitors of haem biosynthesis, haem and cycloheximide on the steady-state expression of mRNAs for ALA synthase and a phenobarbital-inducible cytochrome P-450 (PB1 P-450), in chick embryo hepatocytes in vivo and in primary culture. We found that the mRNAs for ALA synthase and PB1 P-450 were rapidly and simultaneously induced by the porphyrinogenic drugs glutethimide and 2-propyl-2-isopropylacetamide. Inhibitors of haem biosynthesis when administered alone had a small effect on ALA synthase mRNA induction, but in combination with the drugs synergistically increased induction of both ALA synthase mRNA and enzyme activity. However, there were concentrations of inhibitors that increased induction of enzyme activity without increasing mRNA induction. Haem suppressed ALA synthase mRNA induction by drugs by only 50%, whereas induction of ALA synthase enzyme activity was completely suppressed. This suppression of ALA synthase mRNA by haem was blocked by cycloheximide treatment which did not block the induction of ALA synthase mRNA by drugs. In fact, cycloheximide synergistically increased the drug induction of ALA synthase mRNA, suggesting the presence of a labile protein factor which may interact with a haem-responsive element of the ALA synthase gene. Cycloheximide treatment alone did not significantly affect ALA synthase mRNA expression, but induced PB1 P-450 mRNA to a similar extent to that caused by porphyrinogenic drugs, suggesting the presence of a labile repressor which modulates PB1 P-450 gene expression. Basal and drug-inducible PB1 P-450 mRNA levels were unaffected by haem or by inhibitors of haem biosynthesis, indicating that the PB1 P-450 gene is not regulated by haem in chick embryo hepatocytes. Our results indicate that drugs simultaneously induce ALA synthase and PB1 P-450 mRNA expression, and that ALA synthase activity is regulated by haem principally at a post-transcriptional site rather than at the transcriptional level.
Hamilton
J W
JW
Department of Chemistry, Dartmouth College, Hanover, NH 03755.
Bement
W J
WJ
Sinclair
P R
PR
Sinclair
J F
JF
Wetterhahn
K E
KE
D000624:5-Aminolevulinate Synthetase / Q000096:biosynthesis*; D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D003513:Cycloheximide / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D003997:Dicarbethoxydihydrocollidine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004790:Enzyme Induction / Q000187:drug effects; D006418:Heme / Q000096:biosynthesis / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D012333:RNA, Messenger / Q000378:metabolism*
D012333:RNA, Messenger; D006418:Heme; D003997:Dicarbethoxydihydrocollidine; D003577:Cytochrome P-450 Enzyme System; D003513:Cycloheximide; D000624:5-Aminolevulinate Synthetase