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.

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.

The regulation of transferrin receptor and glutathione peroxidase mRNAs synthesis by changes in intracellular iron levels.

The effect of changes of iron availability in the culture medium on the expression of TfR (transferrin receptor) and glutathione peroxidase (GSHPx) genes was investigated in uninduced or induced murine erythroleukemia (Friend) cells of lines 707 and Fw labeled with (3H)uridine for 3 h. The level of the labeled cytoplasmic TfR mRNA exhibited about 2-3-fold increase and the level of the labeled GSHPx mRNA about 2-fold increase in induced Friend 707 cells in comparison with uninduced cells. Raising the levels of intracellular iron by treatment of Friend 707 cells with either hemin, Fe-pyridoxal isonicotinoyl hydrazone (PIH) or diferric transferrin (Tf) resulted in decreased levels of the labeled TfR mRNA. On the other hand, hemin and Fe-PIH caused an increase in the labeled cytoplasmic GSHPx mRNA. Conversely, treatment with PIH or desferrioxamine stimulated synthesis of TfR mRNA and decreased the levels of the labeled GSHPx mRNA. In Fw cells we did not find any difference between the levels of the labeled cytoplasmic TfR mRNA in induced and uninduced cells and the levels of labeled cytoplasmic GSHPx mRNA were only slightly increased by induction. Changes of the intracellular iron pool caused the same effect as in Friend 707 cells.

The role of heme in the regulation of iron uptake during Friend cell differentiation.

The inhibitory effect of hemin on iron uptake and heme synthesis was investigated in Friend erythroleukemia cells. In cells of line 707 the inhibition of iron uptake became more pronounced after the induction of erythroid differentiation by dimethyl sulfoxide. No such rise in sensitivity to hemin was found in cells of line Fw, which are deficient in ferrochelatase activity and do not synthesize hemoglobin after induction of differentiation by sodium butyrate. The possibility that hemin influences mainly the uptake of iron used directly for heme synthesis, and to a lesser extent intracellular storage of iron is discussed.

Control of iron delivery to haemoglobin in erythroid cells.

This paper reviews and reports the results of experiments on the mechanism by which iron is delivered from extracellular transferrin to reticulocyte mitochondria in which haem is synthesized. It is suggested that transferrin donates the iron directly to mitochondria. Transferrin seems to be bound to mitochondria during the process of iron release. When the release of iron from transferrin is blocked by haem, the iron-transferrin complex remains bound to mitochondria so that the total amount of transferrin molecules associated with mitochondria increases in haem-treated reticulocytes. This also leads to an increase in the number of transferrin molecules in the cytosol. In haem-deficient reticulocytes, the rate of dissociation of iron from transferrin is accelerated and the uptake of iron by mitochondria is increased. When the synthesis of haem is inhibited, the non-haem iron in the cytosol (i.e. mainly low-molecular-weight and ferritin iron) comes from mitochondria. Greater amounts of non-haem iron can also be induced in reticulocytes incubated with highly saturated transferrin but, in this case, iron does not seem to be accumulated in mitochondria. These results represent an experimental basis for the elucidation of the excessive non-haem iron accumulation in erythroid cells observed in various clinical conditions.

Effect of hemin and heme synthesis inhibitors on cellular protein synthesis.

Inhibitors of heme synthesis, 2,2'-bipyridyl, isonicotinic acid hydrazide (INH), and D,L-penicillamine markedly inhibited only the synthesis of hemoglobin and had no effect on the synthesis of the bulk of nonhemoglobin proteins in spleen cells of anemic mice. Exogenous hemin stimulated the synthesis of hemoglobin as well as the synthesis of the bulk of nonhemoglobin proteins. However, by further analysis of nonhemoglobin proteins it was possible to detect intermediates of hemoglobin synthesis, globin chains with highly specific radioactivity of L-[4,5-3H]leucine which were eluted together with nonhemoglobin proteins during the chromatography on CM-Sephadex C-50. Protein synthesis in Friend erythroleukemia cells of the Fw line which have the genetic defect of heme synthesis was resistant to D,L-penicillamine and Desferal; 2,2'-bipyridyl had an inhibitory effect. Hemin was without effect on protein synthesis in these neoplastic cells.

Effect of heme on globin messenger RNA synthesis in spleen erythroid cells.

Synthesis of globin mRNA in erythroid spleen cells from anemic mice was measured after in vitro incubation under conditions in which the level of intracellular heme was manipulated. This newly synthesized globin mRNA was isolated by hybridization with globin cDNA covalently bound to cellulose. Isonicotinic acid hydrazide (INH) and penicillamine were used as specific inhibitors of heme synthesis. It has been found that a 120-min incubation of spleen erythroid cells with 5mM INH or 5mM penicillamine reduced [3H] uridine incorporation into globin mRNA by 24% or 36%, respectively. The addition of heme to INH- or penicillamine-treated cells almost completely restored [3H] uridine incorporation into globin mRNA. These results indicate that heme stimulates transcription of processing of globin mRNA.

Effect of heme on globin messenger RNA synthesis.

Erythroid spleen cells from anemic mice were incubated with various inhibitors of heme synthesis. All the effective inhibitors of heme synthesis decreased the incorporation of labelled leucine into protein in spleen erythroid cells. Hemin completely restored protein synthesis to the control levels in isonicotinic acid hydrazide (INH) and penicillamine treated erythroid cells. These two specific inhibitors of heme synthesis were used for the study of the effect of heme on globin mRNA synthesis. Newly synthesized [3H]-uridine labelled globin mRNA was isolated by hybridization to globin cDNA covalently bound to the cellulose column. INH and penicillamine inhibited [3H] uridine incorporation into globin mRNA. The addition of hemin to INH or penicillamine treated cells almost completely restored globin mRNA synthesis. Moreover, hemin alone slightly increased the incorporation of [3H] uridine into globin mRNA. Similar results were obtained in experiments with Friend erythroleukemia cells of the Fw line. These cells are deficient in ferrochelatase enzyme activity and heme synthesis is not significantly increased after induction of differentiation by chemical inducers. The cells induced by butyric acid were incubated for 2 h with or without 100 muM hemin. The incorporation of [3H] uridine into globin mRNA was measured. A 38% increase in globin mRNA synthesis was seen in cells incubated with hemin as compared with cells incubated without hemin. These results seem to indicate that heme affects transcription or processing of globin mRNA precursors. Heme inhibitors also reduce [3H] uridine incorporation into other poly(A)-containing RNA in erythroid spleen cells incubated in vitro. In agreement with these results hemin causes increase of synthesis of some other poly(A)-containing RNA in Friend cells described above.

The onset of hemoglobin synthesis in spleens of irradiated mice after bone marrow transplantation.

Messenger RNA (mRNA) for globin was isolated from spleens of irradiated mice in which erythroid differentiation was induced by a bone marrow graft. The globin mRNA was isolated either by means of sucrose gradients of reticulocyte polysomal RNA or by affinity chromatography of total spleen RNA on poly (U)-sepharose. The globin mRNA was tested in a wheat embryo cell-free system. The appearance of mRNA in the spleen erythroid colonies was correlated with other parameters of erythroid differentiation such as globin synthesis, activity of delta-aminolevulinic acid synthetase and iron uptake. Poly(A) containing mRNA did appear already on the 3rd day after grafting. However, significant translational activity of globin mRNA could be demonstrated only one day later together with the increase in globin synthesis and delta-aminolevulinic acid synthetase and enhanced iron uptake. In the second part of this study mouse spleen cells rich in erythroid elements were incubated with a specific heme synthesis inhibitor (isonicotinic acid hydrazide, INH) and the synthesis of 9 S RNA was estimated. It was found that a 40-minute incubation with INH reduced uridine incorporation into 9 S RNA fraction by about 40%.

Effect of hemin on poly (A)-containing RNA synthesis and transport from nucleus into cytoplasm in Friend cells of the Fw line.

The effect of hemin on the transcription and post-transcriptional events (processing and transport of globin mRNA and other poly(A)-containing RNAs to the cytoplasm) was investigated in murine erythroleukemia Friend cells (MELC) of the Fw line. Uninduced Fw cells or Fw cells induced to differentiation by butyric acid were incubated in vitro without or with exogenous hemin. Short labelling periods (5-20 min) with (3H) uridine were used. The synthesis of globin mRNA and other poly(A)-containing RNAs at the level of their precursors in the nucleus and at the level of mature forms in the cytoplasm was measured. Our results indicate that hemin mainly affects the processing or transport of globin mRNA and other poly(A)-containing RNAs from the nucleus to the cytoplasm. The effect of exogenous hemin is quite different in uninduced and induced Fw cells. Exogenous hemin stimulates accumulation of poly(A)-containing RNA in nuclei of uninduced Fw cells. On the other hand exogenous hemin causes an increase in cytoplasmic globin mRNA and other cytoplasmic poly(A)-containing RNA in Fw cells induced by sodium butyrate.