A different cytochrome P450 form is induced in primary cultures of rat hepatocytes.

Abstract

A 49-kDa protein (P49) was discovered in the primary cultures of rat hepatocytes. P49 cross-reacted with the antibodies against purified P4501IC11 [formerly P-450(M-1)].P49 was located in microsomes and highly induced after plating of isolated hepatocytes on collagen-coated culture dishes. To characterize P49, cDNA clones were screened from a rat liver Agtll expression library. From sequence analysis of the cloned cDNAs, the amino acid sequence of P49 was deduced, and the protein was identified as a previously uncharacterized form of cytochrome P450. P49 consists of 489 amino acids and shows =60% similarity with the members of class IIC subfamily of rat cytochrome P450, such as P45011C11 and P45011C12 [formerly P-450(F-1)]. RNA blot analysis indicates that the mRNA translating P49 was induced "20-to 30-fold at 70 hr in the primary cultures compared with the liver of adult rats. Induction of P49 was not affected by density of the plated cells and the presence or absence of several hormones, serum, or antibiotics in the culture medium. On the other hand, lower induction of P49 was seen when the hepatocytes were cultured on Matrigel-coated plates. Expression of P49 mRNA was low in the liver of adult rats and was not detectable in the livers of 1-and 2-week-old male and female rats. P49 is an additional form of cytochrome P450, which is induced in the primary culture of rat hepatocytes.Primary culture of hepatocytes is a convenient experimental system by which to investigate various liver functions under conditions free from physiological regulations. However, many liver-specific proteins, including cytochrome P450, decrease within a few days in this primary culture. Decrease of liver-specific proteins is mainly due to transcriptional block (1). On the other hand, expressions of a few enzymes, such as heme oxygenase, are highly elevated in primary cultures (2, 3). Synthesis of cytoskeletal proteins, such as a-tubulin and P-actin, are also highly stimulated in primary cultures, but their expressions are lowered when the hepatocytes are plated on certain types of extracellular matrices (4, 5).Hepatic microsomal cytochrome P450 consists of various forms and catalyzes oxidative metabolism of a wide variety of xenobiotic and endogenous substrates. Under conventional culture conditions, cytochrome P450 of the hepatocytes declines rapidly to low levels in a few days of culture (6-8). A few forms of cytochrome P450, such as P45OIA1(formerly P1-450) (9, 10) and P450IIIA1 (formerly P-450p) (11), are induced in the primary cultures with suitable inducers. Recently Guzelian and coworkers (3) developed a culture system using Matrigel for induction of cytochrome P450 in cultured hepatocytes. When rat hepatocytes were cultured on a Matrigel-coated dish and treated with phenobarbital, induced expression of P450IIB1 (formerly P-450b) and P450IIB2 (formerly P-450e) was observed. These are two major phenobarbital-inducible forms of cytochrome P450. However, constitutive forms of cytochrome P450, including P450IIC11 [formerly P...

The kidney cytochrome P-450 2C23 arachidonic acid epoxygenase is upregulated during dietary salt loading

“…In rats the P-450 2C gene subfamily is composed of 8 members (23), 2 of which are sex-specific (P-450s 2C11 and 2C12, ref. 23) and a third, P-450 2C22, that is expressed only in cultured hepatocytes (26). To compare the isoform heterogeneity, the relative abundance of transcripts coding for P-450 2C isoforms, and their transcriptional regulation by dietary salt intake, mRNAs isolated from the livers and kidneys of control and salt-treated rats (2% NaCl as their drinking water for 2 weeks) were electrophoresed and hybridized to 2C isoform-specific oligonucleotides (35-48 mer each, coding for the 3′-end untranslated sequences of each cDNA; ref.…”

Analysis of the Functional Domain of the Rat Liver Mitochondrial Import Receptor Tom20

“…Antibodies against rat monoamine oxidase (MAO), yeast Bmh1p, yeast Kar2p, and yeast Tom70 were generous gifts from Akio Ito (Kyushu University), Masao Sakaguchi (Kyushu University), Masao Tokunaga (Kagoshima University), and Gottfried Schatz (Basel Biocenter), respectively. Antibodies against rat liver microsomal cytochrome P450(M-1), rat liver cytoplasmic hemoprotein H450, and yeast mitochondrial porin were as described (17)(18)(19). Monoclonal antibody against hsp60 (SPA-807) was purchased from Stressgen Biotechnologies Corp.…”

Liver-specific cytochrome P450 CYP2C22 is a direct target of retinoic acid and a retinoic acid-metabolizing enzyme in rat liver

“…The cDNA of the gene now referred to as CYP2C22 was fi rst cloned in 1990 as a cytochrome P450 gene induced after isolated rat hepatocytes were plated on collagen-coated culture dishes ( 24,25 ). Neither of these studies identifi ed RA as a regulator of gene expression or as a substrate for the predicted product.…”