Jitka Borová

A study of intracellular iron metabolism using pyridoxal isonicotinoyl hydrazone and other synthetic chelating agents

Abstract 1. 1. Rabbit reticulocytes with a high level of non-heme radioiron induced by preincubation with isonicotinic acid hydrazide and transferrin-bound 59Fe, were reincubated with various synthetic chelating agents and the amount of radioiron released from the cells was determined. Some substances, especially derivatives of pyridoxal or 2-hydroxybenzaldehyde and isonicotinic acid hydrazide or benzhydrazide, were found to mobilize significantly iron from 59Fe-labeled reticulocytes. The effectiveness of the compounds tested decreases in the following order: pyridoxal isonicotinoyl hydrazone, pyridoxal benzoyl hydrazone, 2-hydroxybenzal isonicotinoyl hydrazone, 2-hydroxybenzal benzoyl hydrazone, pyridoxal-valine Schiff base, pyridoxal. The efficiency of these compounds exceeded the ability of common chelators such as desferrioxamine, 2,2′-bipyridine, nitrolotriacetic acid, etc., to mobilize iron from reticulocytes. 2. 2. Iron mobilization from reticulocytes by pyridoxal isonicotinoyl hydrazone requires ATP to be produced by cells and is completely blocked by low temperature (4°C). Although the effect of desferrioxamine is also prevented by low temperature, modest iron mobilization due to this chelator seems to occur independently of ATP production in reticulocytes. 3. 3. Pyridoxal isonicotinoyl hydrazone mobilizes iron mainly from mitochondria and in part also from ferritin. Although 2,2′-bipyridine seems to enter reticulocyte mitochondria and bind iron there, this chelator is not able to release iron either from mitochondria or from the cells. 4. 4. Reticulocytes with a high level of non-heme radioiron are envisaged as a useful system for testing biological effectiveness of various iron chelators. 5. 5. Pyridoxal isonicotinoyl hydrazone was shown to be an effective in vivo chelator since its administration to mice decreased 59Fe radioactivity in liver, spleen and kidney.

Study of intracellular iron distribution in rabbit reticulocytes with normal and inhibited heme synthesis

Abstract Iron compartments in which iron accumulates during the inhibited heme synthesis after treatment with isonicotinic acid hydrazide were studied in rabbit reticulocytes. A great accumulation of 59Fe radioactivity was found in mitochondria, low molecular weight iron compounds and non-hemoglobin proteins, especially ferritin. In a chase experiment, approximately 50% of the 59Fe radioactivity accumulated in mitochondria and low molecular weight iron compounds was re-utilized for the synthesis of hemoglobin. Although some iron is incorporated into ferritin, it apparently is not utilized for heme synthesis. In control reticulocytes, only traces of 59Fe radioactivity were detected in mitochondria and only a minute amount of 59Fe radioactivity was detected in low molecular weight iron compounds. The release of low molecular weight iron from 59Fe-transferrin occured in intact reticulocytes to a larger extent than in the stroma-free hemolysate. An attempt to establish the possible pathway of iron transport inside the reticulocyte was made. It is suggested that an iron-transferrin complexes enters the reticulocyte cytoplasm, and the majority of released iron is taken up by mitochondria for heme synthesis. When protoporphyrin IX is not available, iron accumulates inside the mitochondria.

Evidence for the presence of free and protein-bound nonhemoglobin heme in rabbit reticulocytes

Abstract In the hemolysate of reticulocytes incubated with 59 Fe or [2– 14 C]glycine a radioactivity is found also in nonhemoglobin heme. About 96% of the radioactivity of nonhemoglobin heme is associated with proteins and approx. 4% is in a free form. Labeled free heme is removed from the reticulocyte hemolysate on the column of Sephadex G-25. Retained heme is eluted from the column of Sephadex G-25 by means of the special eluting solution containing serum albumin. Protein-bound nonhemoglobin heme radioactivity is recovered in hermin isolated from nonhemoglobin proteins separated by means of the chromatography on CM-Sephadex. Isonicotinic acid hydrazide which specifically inhibits heme synthesis prevents the incorporation of 59 Fe into hemoglobin heme. In the hemolysate of cycloheximide incubated reticulocytes the radioactivity in both free and protein-bound nonhemoglobin heme increases essentially. The accumulation of 59 Fe-labeled heme in the nonhemoglobin proteins in reticulocytes incubated with cycloheximide provides evidence for an existence of an increased pool of heme which is not incorporated into hemoglobin or catalase. The finding of the existence of free heme in reticulocytes might contribute to recent theories of regulation of hemoglobin synthesis.

Accumulation of heme in mitochondria from rabbit reticulocytes with inhibited globin synthesis

Incubation of rabbit reticulocytes with cycloheximide and 59Fe bound to transferrin in plasma induces excessive non-hemoglobin 59Fe-labeled heme accumulation in mitochondria. During incubation of these mitochondria in vitro a part of 59Fe-labeled heme is released into the surrounding medium. The addition of globin or bovine serum albumin to the incubation mixture essentially increases the amount of heme released from mitochondria.

The use of exogenous δ-aminolaevulinic acid for the studies of the regulation of haem synthesis in rabbit reticulocytes

Abstract δ-Amino [4- 14 C]laevulinate added to reticulocytes incubated in vitro is incorporated into haem. Exogenous δ-aminolaevulinate restores the incorporation of 59 Fe into haem in reticulocytes which had been treated with isonicotinic acid hydrazide (INH) or penicillamine and were hence unable to synthesize δ-aminolaevulinate. On the other hand, the addition of δ-aminolaevulinate does not restore the incorporation of Fe into reticulocytes incubated with haemin. The inhibition of the incorporation of iron is neither restored by δ-aminolaevulinate in reticulocytes incubated with cycloheximide (which inhibits globin synthesis and thus elevates the free intracellular haem pool). These results suggest that in intact reticulocytes haemin does not inhibit δ-aminolaevulinate synthetase. This conclusion is further supported by the finding that the pattern of incorporation of [2- 14 C]glycine and δ-amino[4- 14 C]-laevulinate into haem differs in reticulocytes incubated with an inhibitor of δ-aminolaevulinate synthetase (INH) and in reticulocytes incubated with haemin and cycloheximide.

Non-transferrin donors of iron for heme synthesis in immature erythroid cells

The mechanism of iron uptake from several iron-containing compounds by transferrin-depleted rabbit reticulocytes and mouse spleen erythroid cells was investigated. Iron complexes of DL-penicillamine, citrate and six different aroyl hydrazones may be utilized by immature erythroid cells for hemoglobin synthesis, although less efficiently than iron from transferrin. HTF-14, a monoclonal antibody against human transferrin, reacts with rabbit transferrin and inhibits iron uptake and heme synthesis by rabbit reticulocytes. HTF-14 had no significant effect on iron uptake and heme synthesis when non-transferrin donors of iron were examined. Ammonium chloride (NH4Cl) increases intracellular pH and blocks the release or utilization of iron from the internalized transferrin. NH4Cl only slightly affected iron incorporation and heme synthesis from non-transferrin donors of iron. Hemin inhibited transferrin iron uptake and heme synthesis, but had a much lesser effect on iron incorporation and heme synthesis from non-transferrin donors of iron. These results allow us to conclude that transferrin-depleted reticulocytes take up iron from all of the examined non-transferrin iron donors without the involvement of the transferrin/transferrin receptor pathway.

Iron and transferrin distribution in reticulocytes incubated with heme synthesis inhibitors

A high level of non-heme iron (either labelled or unlabelled) in mitochondria, ferritin and low-molecular-weight pool of reticulocytes was induced by preincubation with isonicotinic acid hydrazide or penicillamine together with either 59Fe- of 56Fe-labelled transferrin. Addition of apotransferrin during reincubation of 59Fe-labelled reticulocytes was accompanied by the transfer of 59Fe from low-molecular-weight pool to transferrin, which was found in the reticulocyte cytosol both free and bound to a carrier. Similarly, when cells were reincubated with 125I-labelled transferrin, more 125I-labelled radioactivity was found, in both free and carrier-bound transferrin peaks, in reticulocytes with a high level of low-molecular-weight cold iron than in control ones. These results suggest that transferrin enters reticulocytes and takes up iron from low-molecular-weight pool.

The effect of heme on intracellular iron pools during differentiation of friend erythroleukemia cells

The inhibition of cellular iron uptake by hemin described previously in reticulocytes was studied in murine erythroleukemia (Friend) cells that can be induced to differentiate in culture by dimethyl sulfoxide (DMSO). Hemin had no effect on iron uptake into noninduced cells. After the induction by DMSO, hemin inhibited iron uptake into Friend cells and this effect of hemin became more pronounced with the further progress of differentiation. The reduction of cellular iron accumulation was caused mainly by inhibition of iron incorporation into heme, iron uptake into the non-heme pool was little influenced by hemin treatment. Inhibition of heme synthesis by isonicotinic acid hydrazide (INH) caused an accumulation of iron in mitochondria in DMSO-induced cells, but not in uninduced cells. On the basis of these results, a specific system transporting iron to mitochondria induced by DMSO treatment is suggested as a target for the inhibitory action of hemin. In Friend cells of the Fw line which are deficient in ferrochelatase, heme has no effect on iron uptake. The addition of INH to the Fw cells does not enhance the iron accumulation in mitochondria.

Problems of reduced heme synthesis in reticulocytes with impaired globin synthesis

SummaryThe mechanism by which heme synthesis is inhibited in reticulocytes with inhibited globin synthesis is not yet established. Evidence was found that the accumulated free heme does not inhibit the activity of δ-aminolaevulic acidsynthetase. The very early inhibition of heme synthesis may be caused by the effect of uncompleted globin chains or amino acids on the δ-aminolaevulic acidsynthetase or some other enzymes of the porphyrin biosynthetic chain. The accumulated free heme, the presence of which in reticulocytes with inhibited globin synthesis has been indirectly proved may reduce the iron entry to the reticulocytes which in turn can influence the synthesis of heme especially in the later periods of incubation. A feedback inhibitory effect of accumulated free heme at some critical site on the enzymes of the porphyrin biosynthetic chain cannot still be quite excluded.ZusammenfassungDer Mechanismus, mit dem die Häm-Synthese bei Retikulozyten mit gestörter Globulinsynthese gehemmt wird, ist noch nicht geklärt. Es wurden Anzeichen dafür gefunden, daß die akkumulierten freien Häme die Aktivität der δ-Aminolävulinsäuresynthetase nicht behindern. Die sehr frühe Hemmung der Häm-Synthese kann durch die Wirkung unvollständiger Globinketten oder Aminosäuren auf die δ-Aminolävulinsäuresynthetase oder einige andere Enzyme der biosynthetischen Porphyrinkette verursacht werden. Die akkumulierten freien Häme, deren Anwesenheit in Retikulozyten mit gehemmter Globinsynthese indirekt nachgewiesen wurde, können den Eintritt von Eisen in die Retikulozyten reduzieren, was wiederum die Synthese der Häme, besonders in den späteren Inkubationszeiten beeinflussen kann. Eine hemmende Rückkoppelungswirkung akkumulierter freier Häme an irgendeiner kritischen Stelle der Enzyme der biosynthetischen Prophyrinkette kann nicht ganz ausgeschlossen werden.

The effect of heme and iron on haemoglobin A and F synthesis in human cord blood.

Addition of heme to avian erythrocytes and rabbit reticulocytes stimulates globin synthesis. Hammel and Bessman [ 1 ] followed globin synthesis in nuclei of pigeon erythrocytes and proved its stimulation by heme and hypoxia. Bruns and London [2] found stimulated globin synthesis after the addition of heme in reticulocytes of rabbits which were chronically bled and simultaneously fed on iron deficient diet. The mechanism of this effect of heme is not yet completely clear. Heme can increase globin synthesis either by stabilization of polyribosomes, as has been suggested by Waxman and Rabinovitz [3] , or by its attachment to completed globin chains which are released from ribosomes [4]. We have analysed the effect of heme on haemoglobin A and F synthesis in human cord blood. In our further experiments we studied the dependence of the stimulatory effect of heme on the concentration of iron in the incubation medium.