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

Abstract

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.

Oxygen-dependent expression of the erythropoietin gene in rat hepatocytes in vitro

“…The evidence of a role for a short-living haem protein operating as oxygen sensor in the control of EPO gene expression in primary cultures of hepatocytes was found to be less conclusive than reported by Goldberg et al for hepatoma cells [14]. As reported in Hep 3B cells, cycloheximide, dioxoheptanoic acid and desferrioxamine, substances proposed to interfere with the synthesis of such a haem protein [14,35,40] inhibited the hypoxia-induced increases in EPO mRNA levels of isolated hepatocytes (Fig. 6).…”

“…Under this condition, however, total RNA synthesis during the final 2.5 h of incubation, as assessed by [3H]uridine incorporation, was only 2 + 1% (mean + SE, n = 3) of the RNA synthesis in cultures kept at 3 % 02 in the absence of the drug. EPO mRNA accumulation was also inhibited in the presence of desferrioxamine (130 ~tM) (35% reduction), an iron chelator and inhibitor of haem synthesis [35] and dioxoheptanoic acid (2 mM) (79% reduction), an inhibitor of aminolaevulinate dehydratase [40]. During the final 2.5 h of incubation with these two compounds…”

Intracellular Assembly of Inducible NO Synthase Is Limited by Nitric Oxide-mediated Changes in Heme Insertion and Availability

“…This represents the first example of cytokine-induced heme synthesis in a highly differentiated myeloid cell line. Co-induction of heme and hemeprotein synthesis was previously observed during chemical stimulation of cytochrome P-450 expression in chick embryo hepatocytes (37). Thus, like hepatocytes, cytokine-activated RAW 264.7 macrophage cells appear to undergo gene induction to ensure enough heme is made available for insertion into the newly synthesized hemeprotein (iNOS).…”

Neurite Degeneration Induced by Heme Deficiency Mediated via Inhibition of NMDA Receptor-Dependent Extracellular Signal-Regulated Kinase 1/2 Activation

“…For measurement of heme synthesis, cells were incubated with 0.4 Ci of [3,5-3 H]aminolevulinic acid hydrochloride (ALA; 2.6 Ci/mmol; PerkinElmer, Boston, MA) for 24 h. Heme was extracted from the cells by acetone-HCl and diethyl ether. The amount of radioactivity in extracted heme was measured by scintillation counting as described previously (Shedlofsky et al, 1987). Total recovery of radioactivity from all fractions was the same for treated and untreated cells.…”