Apparent lack of correlation between the loss of cytochrome P-450 in hepatic parenchymal cell culture and the stimulation of haem oxygenase activity

Paine, Alan J.; Legg, R.F.

doi:10.1016/0006-291x(78)91589-9

Cited by 100 publications

(19 citation statements)

References 12 publications

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“…Several laboratories have adopted the maintenance of normal levels of cytochrome P450 as a criterion for liver parenchymal cell viability [29,30]. Rat hepatocytes cultured for 24 h lose 70% of their cytochrome P450 [31,32], while in bovine adrenal zona glomerulosa cells the loss of capacity to synthesize aldosterone accompanies the decline in cytochrome P450 following isolation [33,34]. This decrease can be prevented by supplementing the culture medium with a variety of nutrients, growth factors and hormones [35,36], or by the incorporation of hyperphysiological concentrations of nicotinamide or pyridine derivatives (metapyrone) in the culture medium [37,38].…”

Section: Cell Isolationmentioning

confidence: 99%

Primary culture, cellular stress and differentiated function

Wolffe

Tata

1984

FEBS Letters

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Isolation of specialized cell types for the analysis of tissue‐specific gene function often results in loss of the differentiated phenotype. Examples of this type of phenotypic change following tissue disaggregation are reviewed together with possible explanations. Close similarities between the effects of cell isolation with those of other cellular stresses such as heat or anoxia point to common biochemical mechanisms being involved. This suggests that the study of freshly isolated cells will contribute significantly to out understanding of the nature of cellular stress and its consequences for the maintenance of phenotype and induction of tissue specific gene expression.

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Section: Cell Isolationmentioning

confidence: 99%

Primary culture, cellular stress and differentiated function

Wolffe

Tata

1984

FEBS Letters

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“…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).…”

mentioning

confidence: 99%

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

Emi

Chijiiwa

Omura

1990

Proc. Natl. Acad. Sci. U.S.A.

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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...

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“…Cytochrome P450 content of intact liver, isolated cells and hepatocytes cultured for 24h, was determined by recording on a Unicam SP.8-100 spectrophotometer (1 cm light path) the difference spectrum of reduced homogenate plus CO versus reduced 0306-3283/79/11/461-03 $1.50/1 homogenate, by taking the precautions previously described (Paine & Legg, 1978). For this determination the samples used were homogenized in 20 % (v/v) glycerol in 0.1 M-sodium phosphate buffer, pH7.4, containing 1 mM-EDTA and 0.2% (w/v) emulgen (Sinclair et al, 1979) by ten up-and-down strokes of a glass homogenizer fitted with a Teflon pestle, with a clearance of 0.5mm and driven at 2000 rev./min.…”

Section: Isolation Ofhepatocytesmentioning

confidence: 99%

“…The primary culture of hepatic parenchymal cells results in the spontaneous loss of 70 % of their cytochrome P-450 during the first 24h (Guzelian et al, 1977;Paine & Legg, 1978). This spontaneous loss can be prevented by the inclusion of high unphysiological concentrations of nicotinamide in the culture medium (Paine et al, 1979a).…”

mentioning

confidence: 99%

Relationship between the ability of nicotinamide to maintain nicotinamide-adenine dinucleotide in rat liver cell culture and its effect on cytochrome P-450

Paine¹,

Hockin²,

Legg³

1979

Biochemical Journal

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Rat hepatocytes cultured for 24h lose 60% of their NAD content. Treatment with nicotinamide prevents the loss of NAD as well as the previously reported loss of cytochrome P.450, suggesting a possible causal relationship. However, isonicotinamide also prevents the loss of cytochrome P-450, but does not increase the concentration of NAD, demonstrating that the ability of nicotinamide to maintain cytochrome P-450 is not apparently related to its effect on the NAD content of cultured hepatocytes.

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Apparent lack of correlation between the loss of cytochrome P-450 in hepatic parenchymal cell culture and the stimulation of haem oxygenase activity

Cited by 100 publications

References 12 publications

Primary culture, cellular stress and differentiated function

Primary culture, cellular stress and differentiated function

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

Relationship between the ability of nicotinamide to maintain nicotinamide-adenine dinucleotide in rat liver cell culture and its effect on cytochrome P-450

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