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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 P450IIC11 [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 lambda gt11 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 approximately 60% similarity with the members of class IIC subfamily of rat cytochrome P450, such as P450IIC11 and P450IIC12 [formerly P-450(F-1)]. RNA blot analysis indicates that the mRNA translating P49 was induced approximately 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.

SUBMITTER: Emi Y

PROVIDER: S-EPMC55250 | biostudies-other | 1990 Dec

REPOSITORIES: biostudies-other

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

Emi Y Y Chijiiwa C C Omura T T

Proceedings of the National Academy of Sciences of the United States of America 19901201 24

A 49-kDa protein (P49) was discovered in the primary cultures of rat hepatocytes. P49 cross-reacted with the antibodies against purified P450IIC11 [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 lambda gt11 expression library. From sequence analysis of the cloned cDNAs, the amino acid sequence of P49 was deduced, and the protein was iden ...[more]

PMID: 2263625

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Project description:UnlabelledAngiotensin II (AT-II) is a pro-fibrotic compound that acts via membrane-bound receptors (AT-1R/AT-2R) and thereby activates hepatic stellate cells (HSCs). AT-II receptor blockers (ARBs) are thus important candidates in the treatment of liver fibrosis. However, multiple case reports suggest that AT-1R blockers may induce hepatocyte injury. Therefore, we investigated the effect of AT-II and its receptor blockers on cytokine-, oxidative stress- and bile salt-induced cell death in hepatocytes. Primary rat hepatocytes were exposed to TNF-α/Actinomycin D, the ROS-generating agent menadione or the bile salts: glycochenodeoxycholic acid (GCDCA) and tauro-lithocholic acid-3 sulfate (TLCS), to induce apoptosis. AT-II (100 nmol/L) was added 10 minutes prior to the cell death-inducing agent. AT-1R antagonists (Sartans) and the AT-2R antagonist PD123319 were used at 1 µmol/L. Apoptosis (caspase-3 activity, acridine orange staining) and necrosis (Sytox green staining) were quantified. Expression of CHOP (marker for ER stress) and AT-II receptor mRNAs were quantified by Q-PCR. AT-II dose-dependently reduced GCDCA-induced apoptosis of hepatocytes (-50%, p<0.05) without inducing necrosis. In addition, AT-II reduced TLCS-induced apoptosis of hepatocytes (-50%, p<0.05). However, AT-II did not suppress TNF/Act-D and menadione-induced apoptosis. Only the AT-1R antagonists abolished the protective effect of AT-II against GCDCA-induced apoptosis. AT-II increased phosphorylation of ERK and a significant reversal of the protective effect of AT-II was observed when signaling kinases, including ERK, were inhibited. Moreover, AT-II prevented the GCDCA-induced expression of CHOP (the marker of the ER-mediated apoptosis).ConclusionAngiotensin II protects hepatocytes from bile salt-induced apoptosis through a combined activation of PI3-kinase, MAPKs, PKC pathways and inhibition of bile salt-induced ER stress. Our results suggest a mechanism for the observed hepatocyte-toxicity of Sartans (angiotensin receptor blockers, ARBs) in some patients with chronic liver injury.

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

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

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