Expression of multiple forms of cytochrome P450 mRNAs in primary cultures of rat hepatocytes maintained on matrigel.
Freshly isolated rat hepatocytes rapidly lose their cytochrome P450 (P450) proteins and mRNAs, with no evidence of subsequent restoration, after placement into traditional systems of primary culture on type I collagen. We examined the patterns of expression of 10 constitutively expressed P450 mRNAs in rat hepatocytes cultured for up to 5 days on a matrix of Matrigel, a reconstituted basement membrane that allows inducible expression of some P450s, and compared these patterns with those seen in hepatocytes cultured on type I collagen (Vitrogen). mRNA for each P450 was detected on Northern blots in samples prepared from freshly isolated male rat hepatocytes, and the amount of each mRNA decreased markedly during the first 2 days of culture in cells maintained on either matrix (decreases of 53-97% on Matrigel and 62 to > 99% on Vitrogen), in contrast to the level of NADPH-P450 oxidoreductase mRNA, which increased during this interval. On subsequent days, hepatocytes cultured on Matrigel expressed the individual P450 mRNAs in one of the following four patterns. 1) P450 1A2, 2A2, 2E1, and 3A1/2 mRNAs remained low throughout the 5-day culture period. 2) 2A1 mRNA increased between days 2 and 4 but then decreased on day 5. 3) 2B1/2, 2C6, 2C7, and 4A1 mRNAs increased continually between days 2 and 5, 4) The total mRNAs detected with a 2D probe remained at constant levels between culture days 2 and 5. Our results show that rat hepatocytes cultured on Matrigel in serum-free medium spontaneously reexpress the mRNAs for several constitutive P450s in form-specific patterns, and they suggest that the Matrigel culture system will be useful for identifying the underlying regulatory mechanisms.
Kocarek
T A
TA
Department of Medicine, Medical College of Virginia, Richmond 23298.
Schuetz
E G
EG
Guzelian
P S
PS
D000818:Animals; D001345:Autoradiography; D001485:Basement Membrane / Q000737:chemistry; D015152:Blotting, Northern; D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry*; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics*; D004338:Drug Combinations; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D007797:Laminin / Q000737:chemistry*; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D011509:Proteoglycans / Q000737:chemistry*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; D012333:RNA, Messenger; C059485:matrigel; D003094:Collagen; D003577:Cytochrome P-450 Enzyme System