scholarly journals Haem synthesis from exogenous 5-aminolaevulinate in cultured chick-embryo hepatocytes. Effects of inducers of cytochromes P-450

1987 ◽  
Vol 248 (1) ◽  
pp. 229-236 ◽  
Author(s):  
S I Shedlofsky ◽  
P R Sinclair ◽  
H L Bonkovsky ◽  
J F Healey ◽  
A T Swim ◽  
...  
Keyword(s):  
Chick Embryo ◽  
Iron Chelator ◽  
Cellular Concentration ◽  
Haem Biosynthesis ◽  
Cytochrome P 450 ◽  
In Cells

The effects of inducers of cytochrome P-450 on haem biosynthesis from 5-aminolaevulinate were examined by using cultured chick-embryo hepatocytes. Cultures treated with either 2-propyl-2-isopropylacetamide or 3-methylcholanthrene contained increased amounts of cytochrome P-450 and haem. After treatment for 3 h with 5-amino[4-14C]laevulinate, the relative amounts of radioactivity accumulating as haem corresponded to the relative amounts of total cellular haem, but not to increases in the amounts of cytochrome P-450. Treatment with 5-aminolaevulinate did not alter cellular haem or cytochrome P-450 concentrations in either control or drug-treated cultures. The mechanism of the enhanced accumulation of radioactivity in haem was investigated. Although 2-propyl-2-isopropylacetamide enhanced the uptake of 5-aminolaevulinate and increased the cellular concentration of porphobilinogen 1.5-fold, these changes did not account for the increases in haem radioactivity. The inducing drugs had no effect on the rates of degradation of radioactive haem, but appeared to enhance conversion of protoporphyrin into haem. This latter effect was shown by: (1) a decreased accumulation of protoporphyrin from 5-aminolaevulinate in cells treated with inducers, and (2) complete prevention of this decrease if the iron chelator desferrioxamine was present. We conclude that inducers of cytochrome P-450 may increase haem synthesis not only by increasing activity of 5-aminolaevulinate synthase, but also by increasing conversion of protoporphyrin into haem.

scholarly journals Role of haem in the induction of cytochrome P-450 by phenobarbitone. Studies in chick embryos in ovo and in cultured chick embryo hepatocytes

1981 ◽  
Vol 198 (2) ◽  
pp. 321-329 ◽  
Author(s):  
U Giger ◽  
U A Meyer
Keyword(s):  
Chick Embryo ◽  
Inhibitory Effect ◽  
Chick Embryos ◽  
In Ovo ◽  
Haem Biosynthesis ◽  
Rate Limiting ◽  
Cytochrome P 450 ◽  
Hepatic Cytochrome

The role of haem synthesis during induction of hepatic cytochrome P-450 haemoproteins was studied in chick embryo in ovo and in chick embryos hepatocytes cultured under chemically defined conditions. 1. Phenobarbitone caused a prompt increase in the activity of 5-aminolaevulinate synthase, the rate-limiting enzyme of haem biosynthesis, and in the concentration of cytochrome P-450. This induction response occurred without measurable initial destruction of the haem moiety of cytochrome P-450. 2. When intracellular haem availability was enhanced by exogenous haem or 5-aminolaevulinate, phenobarbitone-medicated induction of cytochrome P-450 was not affected in spite of the well known repression of 5-aminolaevulinate synthase by haem. These data are consistent with the concept that haem does not regulate the synthesis of cytochrome P-450 haemoproteins. 3. Acetate inhibited haem biosynthesis at the level of 5-aminolaevulinate formation. When intracellular haem availability was diminished by treatment with acetate, phenobarbitone-medicated induction was decreased. 4. This inhibitory effect of acetate on cytochrome P-450 induction was reversed by exogenous haem or its precursor 5-aminolaevulinate. These data suggest that inhibition of haem biosynthesis does not decrease synthesis of apo-cytochrome P-450. Moreover, they indicate that exogenous haem can be incorporated into newly formed aop-cytochrome P-450.

scholarly journals Decrease in hepatic cytochrome P-450 by cobalt. Evidence for a role of cobalt protoporphyrin

1982 ◽  
Vol 204 (1) ◽  
pp. 103-109 ◽  
Author(s):  
J F Sinclair ◽  
P R Sinclair ◽  
J F Healey ◽  
E L Smith ◽  
H L Bonkowsky
Keyword(s):  
Chick Embryo ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Cobalt Protoporphyrin ◽  
Cytochrome P 450 ◽  
Hepatic Cytochrome

Exposure of cultured chick-embryo hepatocytes to increasing concentrations of CoCl2 in the presence of allylisopropylacetamide results in formation of cobalt protoporphyrin, with a reciprocal decrease in haem and cytochrome P-450. Treatment of rats with CoCl2 (84 mumol/kg) and 5-aminolaevulinate (0.2 mmol/kg) also results in formation of cobalt protoporphyrin and a decrease in cytochrome P-450 in the liver. Hepatic microsomal fractions from rats treated with phenobarbital, CoCl2 and 5-aminolaevulinate were analysed by polyacrylamide gel electrophoresis. Cobalt protoporphyrin was associated mainly with proteins of 50000-53000 mol.wt. The results suggest that the formation of cobalt protoporphyrin occurred at the expense of the synthesis of haem, leading to a decrease in cytochrome P-450. Furthermore, the cobalt protoporphyrin that was formed may itself have been incorporated into apocytochrome P-450.

scholarly journals Agonist-induced Ca2+ influx in human neutrophils is secondary to the emptying of intracellular calcium stores

1991 ◽  
Vol 277 (1) ◽  
pp. 73-79 ◽  
Author(s):  
M Montero ◽  
J Alvarez ◽  
J Garcia-Sancho
Keyword(s):  
Plasma Membrane ◽  
Intracellular Calcium ◽  
Time Course ◽  
Human Neutrophils ◽  
Calcium Stores ◽  
Free Medium ◽  
Prolonged Incubation ◽  
Intracellular Calcium Stores ◽  
Cytochrome P 450 ◽  
In Cells

Emptying of the intracellular calcium stores of human neutrophils, by prolonged incubation in Ca(2+)-free medium, by treatment with low concentrations of the Ca2+ inophore ionomycin, or by activation with cell agonists, increased the plasma-membrane permeability to Ca2+ and Mn2+. The chemotactic peptide formylmethionyl-leucyl-phenylalanine and the natural agonists platelet-activating factor and leukotriene B4 released different amounts of calcium from the stores and induced Ca2+ (Mn2+) uptake, the rate of which correlated inversely with the amount of calcium left in the stores. The increased Mn2+ uptake induced by these agonists was persistent in cells incubated in Ca(2+)-free medium, but returned to basal levels in cells incubated in Ca(2+)-containing medium, with the same time course as the refilling of the calcium stores. The calcium-stores-regulated Mn2+ influx, including that induced by agonists, was prevented by cytochrome P-450 inhibitors. We propose that agonist-induced Ca2+ (Mn2+) influx in human neutrophils is secondary to the emptying of the intracellular stores which, in turn, activates plasma-membrane Ca2+ channels by a mechanism involving microsomal cytochrome P-450, similar to that described previously in thymocytes [Alvarez, Montero & Garcia-Sancho (1991) Biochem. J. 274, 193-197].

scholarly journals Regulation of hepatic haem metabolism. Disparate mechanisms of induction of haem oxygenase by drugs and metals

1988 ◽  
Vol 250 (1) ◽  
pp. 189-196 ◽  
Author(s):  
B C Lincoln ◽  
J F Healey ◽  
H L Bonkovsky
Keyword(s):  
Chick Embryo ◽  
Primary Culture ◽  
In Ovo ◽  
Oxygenase Activity ◽  
Haem Oxygenase ◽  
Cytochrome P 450

We studied drug- and metal-mediated increases in activity of haem oxygenase, the rate-controlling enzyme for haem breakdown, in chick-embryo hepatocytes in ovo and in primary culture. Phenobarbitone and phenobarbitone-like drugs (glutethimide, mephenytoin), which are known to increase concentrations of an isoform of cytochrome P-450 in chick-embryo hepatocytes, were found to increase activities of haem oxygenase as well. In contrast, 20-methylcholanthrene, which increases the concentration of a different isoform of cytochrome P-450, had no effect on activity of haem oxygenase. Inhibitors of haem synthesis, 4,6-dioxoheptanoic acid or desferrioxamine, prevented drug-mediated induction of both cytochrome P-450 and haem oxygenase in embryo hepatocytes in ovo or in culture. Addition of haem restored induction of both enzymes. These results are interpreted to indicate that phenobarbitone and its congeners induce haem oxygenase by increasing hepatic haem formation. In contrast, increases in haem oxygenase activity by metals such as cobalt, cadmium and iron were not dependent on increased haem synthesis and were not inhibited by 4,6-dioxoheptanoic acid. We conclude that (1) induction of hepatic haem oxygenase activity by phenobarbitone-type drugs is due to increased haem formation, and (2) induction of haem oxygenase by drugs and metals occurs by different mechanisms.

scholarly journals Uroporphyrin accumulation produced by halogenated biphenyls in chick-embryo hepatocytes. Reversal of the accumulation by piperonyl butoxide

1986 ◽  
Vol 237 (1) ◽  
pp. 63-71 ◽  
Author(s):  
P R Sinclair ◽  
W J Bement ◽  
H L Bonkovsky ◽  
R W Lambrecht ◽  
J E Frezza ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Chick Embryo ◽  
De Novo ◽  
Covalent Binding ◽  
Piperonyl Butoxide ◽  
Uroporphyrinogen Decarboxylase ◽  
Drug Induced ◽  
Cytochrome P 450 ◽  
Inhibition Of Metabolism ◽  
Methylenedioxyphenyl Compounds

Cultures of chick-embryo hepatocytes were used to study the mechanism by which 3,4,3′,4′-tetrachlorobiphenyl and 2,4,5,3′,4′-pentabromobiphenyl cause accumulation of uroporphyrin. In a previous paper, an isoenzyme of cytochrome P-450 induced by 3-methylcholanthrene had been implicated in this process [Sinclair, Bement, Bonkovsky & Sinclair (1984) Biochem. J. 222, 737-748]. Cells treated with 3,4,3′,4′-tetrachlorobiphenyl and 5-aminolaevulinate accumulated uroporphyrin and heptacarboxyporphyrin, whereas similarly treated cells accumulated protoporphyrin immediately after piperonyl butoxide was added. Piperonyl butoxide also restored haem synthesis as detected by incorporation of radioactive 5-aminolaevulinate into haem, and decrease in drug-induced 5-aminolaevulinate synthase activity. The restoration of synthesis of protoporphyrin and haem by piperonyl butoxide was not affected by addition of cycloheximide, indicating recovery was probably not due to protein synthesis de novo. Piperonyl butoxide also reversed uroporphyrin accumulation caused by 3,4,5,3′,4′,5′-hexachlorobiphenyl, mixtures of other halogenated biphenyls, lindane, parathion, nifedipine and verapamil. The effect of piperonyl butoxide was probably not due to inhibition of metabolism of these compounds, since the hexachlorobiphenyl was scarcely metabolized. Other methylenedioxyphenyl compounds, as well as ellipticine and acetylaminofluorene, also reversed the uroporphyrin accumulation caused by 3,4,3′,4′-tetrachlorobiphenyl. SKF-525A (2-dimethylaminoethyl-2,2-diphenyl valerate) did not reverse the uroporphyrin accumulation caused by the halogenated biphenyls, but did reverse that caused by phenobarbital and propylisopropylacetamide. We conclude that the mechanism of the uroporphyrin accumulation cannot be due to covalent binding of activated metabolites of halogenated compounds to uroporphyrinogen decarboxylase.

scholarly journals Underhydroxylated minor cartilage collagen precursors cannot form stable triple helices

1988 ◽  
Vol 250 (1) ◽  
pp. 65-70 ◽  
Author(s):  
C C Clark ◽  
C F Richards
Keyword(s):  
Chick Embryo ◽  
Triple Helix ◽  
Iron Chelator ◽  
Prolyl Hydroxylase ◽  
Type Ii ◽  
Native Structure ◽  
Lysyl Hydroxylase ◽  
Free Cells ◽  
Triple Helices ◽  
Matrix Free

Matrix-free cells from chick-embryo sterna were incubated with various concentrations of 2,2′-bipyridyl, an iron chelator that inhibits prolyl hydroxylase and lysyl hydroxylase. At concentrations in the region of 0.1 mM, significant effects on cartilage collagen hydroxylation and secretion were observed. When the underhydroxylated collagens were subsequently digested with chymotrypsin or chymotrypsin plus trypsin at 4 degrees C for 15 min, the minor cartilage collagen precursors (namely types IX and XI) were extensively degraded; type II procollagen was only partially susceptible and was converted into underhydroxylated collagen. The results demonstrate that there were significant differences in triple-helix stability among cartilage collagens such that the underhydroxylated minor collagen precursors were unable to attain a native structure under conditions where type II procollagen was successful.

scholarly journals Hydroxylation of salicylate by microsomal fractions and cytochrome P-450. Lack of production of 2,3-dihydroxybenzoate unless hydroxyl radical formation is permitted

1991 ◽  
Vol 276 (3) ◽  
pp. 753-757 ◽  
Author(s):  
M Ingelman-Sundberg ◽  
H Kaur ◽  
Y Terelius ◽  
J O Persson ◽  
B Halliwell
Keyword(s):  
Hydroxyl Radical ◽  
Hydroxyl Radicals ◽  
Liver Microsomes ◽  
Iron Chelator ◽  
Radical Formation ◽  
Rabbit Liver ◽  
Nadph Cytochrome ◽  
Substrate Hydroxylation ◽  
Cytochrome P 450

Attack by hydroxyl radicals (.OH) upon salicylate (2-hydroxybenzoate) leads to formation of both 2,3-dihydroxybenzoate (2,3-DHB) and 2,5-dihydroxybenzoate (gentisate, 2,5-DHB). It has been suggested that formation of 2,3-DHB from salicylate is a means of monitoring .OH formation. Production of 2,3-DHB and 2,5-DHB by liver microsomal fractions and isoforms of cytochrome P-450 was investigated. Liver microsomes prepared from variously treated rats and rabbits catalysed the formation of 2,5-DHB but not 2,3-DHB. Formation of 2,5-DHB was inhibited by CO, metyrapone and SKF-525A, but not by the .OH scavengers mannitol and formate or by the iron chelator desferrioxamine. Purified P-450s IIE1, IIB4 or IA2 from rabbit liver microsomes, reconstituted together with NADPH-cytochrome P-450 reductase, led to formation of equal amounts of 2,3-DHB and 2,5-DHB in reactions that were almost completely inhibited by mannitol or formate. Addition of Fe3+/EDTA either to microsomes or to membranes containing reconstituted P-450 caused formation of approximately equal amounts of 2,3-DHB and 2,5-DHB, consistent with an .OH-dependent attack on salicylate. The data indicate that the microsomal P-450 system catalyses hydroxylation of salicylate to 2,5-DHB, but not formation of 2,3-DHB. Hence measurement of 2,3-DHB might provide a means of monitoring .OH formation. Care must be taken in studies of substrate hydroxylation by microsomes or reconstituted P-450 systems to avoid artefacts resulting from .OH generation.

Induction of cytochrome P-450 in a selective subpopulation of hepatocytes

1978 ◽  
Vol 234 (3) ◽  
pp. C102-C109 ◽  
Author(s):  
J. J. Gumucio ◽  
L. J. DeMason ◽  
D. L. Miller ◽  
S. O. Krezoski ◽  
M. Keener
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Inductive Effect ◽  
Specific Activity ◽  
Smooth Endoplasmic Reticulum ◽  
Cell Subpopulation ◽  
Continuous Density ◽  
Liver Cytochrome ◽  
Cytochrome P 450 ◽  
In Cells

The objective of this study was to determine whether the inductive effect of phenobarbital (PB) on liver cytochrome P-450 was the result of the action of this drug on all or some hepatocytes. For this purpose, a light (cell band I) and a heavy (cell band II) subpopulation of hepatocytes were separated from rat liver in a continuous density gradient. To determine the location of these hepatocytes in tissue, [14C]bromobenzene, which binds covalently to centrilobular hepatocytes, was administered. The specific activity (14C dpm/mg protein) was greater in cells of band I than in cells of band II, suggesting a predominant contribution of centrilobular hepatocytes to the lighter cell band. Microsomes were separated from each cell subpopulation after 3 days of PB administration and cytochrome P-450 was measured. Although a fivefold increment in cytochrome P-450 content of light hepatocytes was noted, the content of heavy hepatocytes was similar to that of the respective subpopulation in controls. Concomitantly, PB administered for 3 days induced the smooth endoplasmic reticulum of centrilobular hepatocytes only, as revealed by electron microscopy of whole tissue. These results indicated that PB induces cytochrome P-450 in a selective subpopulation of hepatocytes, most likely located near the terminal hepatic venule.

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