Haem control in experimental porphyria. The effect of haemin on the induction of δ-aminolaevulinate synthase in isolated chick-embryo liver cells

Abstract

2-Allyl-2-isopropylacetamide-mediated induction of hepatic porphyria was studied in isolated chick-embryo liver cells. Increased δ-aminolaevulinate synthase activity occurred within 1 h of induction and continued to increase for 8 h. Protoporphyrins synthesized during this time accumulated to a concentration 1-fold greater than that in the control. Removal of 2-allyl-2-isopropylacetamide from the cells by washing at 3 h immediately inhibited further increases in δ-aminolaevulinate synthase synthesis. However substitution of 2-allyl-2-isopropylacetamide at 3 h by deferoxamine methane-sulphonate, an inhibitor of haem synthesis, allowed continued δ-aminolaevulinate synthase induction at an unaltered rate, even though this agent did not, by itself, induce enzyme synthesis. Exogenously added haemin was shown completely to inhibit 2-allyl-2-isopropylacetamide-mediated δ-aminolaevulinate synthase induction at concentrations as low as 20 nM, a value that is less than the reported physiological one. The duration of inhibition was dependent on the concentration of added haemin and was followed by period of δ-aminolaevulinate synthase synthesis at a rate similar to that of the control. These data are consistent with the hypothesis that δ-aminolaevulinate synthase synthesis is regulated by the concentration of intracellular haem and that induction is initiated by 2-allyl-2-isopropylacetamide-mediated destruction of haem. Induction of δ-aminolaevulinate synthase was shown to be dependent on both RNA and protein synthesis, and a study of the comparative effects of cordycepin, cycloheximide and haem has shown that, at haemin concentrations up to 50 nM, the inhibition of δ-aminolaevulinate synthase synthesis followed kinetics similar to the effect of cordycepin, with no synergism between cordycepin and 50nM-haemin. However, at a haemin concentration of 2 μM, the inhibition of δ-aminolaevulinate synthase synthesis followed similar kinetics to the effect of cycloheximide. These data demonstrate the control of δ-aminolaevulinate synthase synthesis by low concentrations of haemin and suggests that the primary effect of haemin is at the level of transcription.