Heme Synthesis In Reticulocytes Research Articles

Nitrogen Monoxide Inhibits Heme Synthesis In Reticulocytes

AbstractAbstract 4252Anemia of chronic disease (ACD) is a condition that often manifests in patients with chronic immune activation due to chronic infections, autoimmune disorders, cancer and other diseases. The pathogenesis of ACD is complex and involves inefficient erythropoietin production, immune-mediated inhibition of erythropoiesis, and retention of iron in hemoglobin-processing macrophages. During their development, erythroid cells are closely associated with macrophages. In inflammatory conditions, activated macrophages generate large quantities of the gaseous molecule, nitric oxide (NO), which has numerous effects on iron metabolism. In this study, we explored the possibility that NO affects iron metabolism in erythroid cells. We treated reticulocytes with the NO donors, sodium nitroprusside (SNP) and S-Nitroso-N-acetyl-D,L-penicillamine (SNAP). We show that NO inhibits 59Fe incorporation from 59Fe-transferrin into reticulocytes and their heme. Significantly, 5-aminolevulinic acid (ALA, the product of ALA synthase, which catalyzes the first step of heme synthesis) reversed the SNP-mediated decrease in 59Fe incorporation into heme but not the cellular 59Fe uptake. In addition, SNP treatment led to an increase in eIF2α phosphorylation (which is known to occur in heme-deficient cells) and decreased globin translation. Importantly, the addition of ALA to SNP-treated reticulocytes prevented the effect of SNP on eIF2α phosphorylation and reversed globin synthesis inhibition. This indicates that in SNP treated reticulocytes, the phosphorylation of eIF2α and inhibition of globin synthesis occur indirectly, via NO's effect on erythroid-specific ALA synthase (ALA-S2). These results led us to conclude that NO has two distinct effects on reticulocytes, namely: a decrease in ALA-S2 activity and a decrease in transferrin-mediated iron uptake. The profound impact of NO on heme synthesis, iron uptake and globin translation in reticulocytes raises the possibility that NO production by macrophages could also contribute to ACD. Disclosures:No relevant conflicts of interest to declare.

Mechanisms of iron transport into rat erythroid cells

Three mechanisms of iron uptake by rat erythroid cells were identified, two with non-transferrin-bound iron (NTBI) and one with transferrin-bound iron (Fe-Tf). Uptake of NTBI occurred by a high affinity mechanism (Km approximately 0.1 μM). Activity of the high affinity mechanism was maximal in sucrose solution and of the low affinity mechanism in KCl solution. Both were inhibited by NaCl and by certain ion transport inhibitors, but they differed in their sensitivity to the various inhibitors. Fe-Tf uptake was also of high affinity (Km 0.1 μM). All the transport mechanisms show higher activity in reticulocytes than in mature erythrocytes, and all could provide iron for heme synthesis in reticulocytes. The results demonstrate certain conditions which should be followed in order to study high affinity transport of NTBI. These include use of a low packed cell volume in the incubation mixture, low iron concentrations (0.01–1.0 μM), short incubation times (up to 20 min), and low osmolality (approximately 200 mOsm/kg) during incubation with the NTBI and subsequent washing of the cells. J. Cell. Physiol. 186:193–200, 2001. © 2001 Wiley-Liss, Inc.

Acquisition of iron from transferrin regulates reticulocyte heme synthesis.

Fe-salicylaldehyde isonicotinoylhydrazone (SIH), which can donate iron to reticulocytes without transferrin as a mediator, has been utilized to test the hypothesis that the rate of iron uptake from transferrin limits the rate of heme synthesis in erythroid cells. Reticulocytes take up 59Fe from [59Fe]SIH and incorporate it into heme to a much greater extent than from saturating concentrations of [59Fe]transferrin. Also, Fe-SIH stimulates [2-14C]glycine into heme when compared to the incorporation observed with saturating levels of Fe-transferrin. In addition, delta-aminolevulinic acid does not stimulate 59Fe incorporation into heme from either [59Fe]transferrin or [59Fe]SIH but does reverse the inhibition of 59Fe incorporation into heme caused by isoniazid, an inhibitor of delta-aminolevulinic acid synthase. Taken together, these results suggest the hypothesis that some step(s) in the pathway of iron from extracellular transferrin to intracellular protoporphyrin limits the overall rate of heme synthesis in reticulocytes.

Regulation of Heme Synthesis in Erythroid Cells: Hemin Inhibits Transferrin Iron Utilization but Not Protoporphyrin Synthesis

The inhibition of delta-aminolevulinic acid (ALA) synthase activity by heme is commonly thought to regulate the overall rate of heme synthesis in erythroid cells. However, since heme inhibits erythroid cell uptake of iron from transferrin, we have tested the hypothesis that in reticulocytes heme regulates its own synthesis by controlling the cellular acquisition of iron from transferrin rather than by controlling the synthesis of ALA. We found that hemin added to reticulocytes in vitro inhibits not only the total cell incorporation of 59Fe from transferrin but also the incorporation of [2-14C]-glycine and transferrin-bound 59Fe into heme. However, hemin did not inhibit [2-14C]-glycine incorporation into protoporphyrin. Furthermore, cycloheximide, which increases the level of non-hemoglobin heme in reticulocytes, also inhibited [2-14C]-glycine into heme but not into protoporphyrin. With high concentrations of ferric pyridoxal benzoylhydrazone (Fe-PBH), which, independent of transferrin and transferrin receptors, can be used as a source of iron for heme synthesis in reticulocytes, significantly more iron is incorporated into heme than from saturating concentrations of Fe-transferrin. This suggests that some step (or steps) in the pathway of iron from extracellular transferrin to protoporphyrin limits the overall rate of heme synthesis in reticulocytes. In addition, hemin in concentrations that inhibit the utilization of transferrin-bound iron for heme synthesis has no effect on the incorporation of iron from Fe-PBH into heme. Our results indicate that in reticulocytes heme inhibits and controls the utilization of iron from transferrin but has no effect on the enzymes of porphyrin biosynthesis and ferrochelatase. This mode of regulation of heme synthesis may be a specific characteristic of the hemoglobin biosynthetic pathway.

Regulation of heme synthesis in erythroid cells: hemin inhibits transferrin iron utilization but not protoporphyrin synthesis

The inhibition of delta-aminolevulinic acid (ALA) synthase activity by heme is commonly thought to regulate the overall rate of heme synthesis in erythroid cells. However, since heme inhibits erythroid cell uptake of iron from transferrin, we have tested the hypothesis that in reticulocytes heme regulates its own synthesis by controlling the cellular acquisition of iron from transferrin rather than by controlling the synthesis of ALA. We found that hemin added to reticulocytes in vitro inhibits not only the total cell incorporation of 59Fe from transferrin but also the incorporation of [2–14C]-glycine and transferrin-bound 59Fe into heme. However, hemin did not inhibit [2 –14C]-glycine incorporation into protoporphyrin. Furthermore, cycloheximide, which increases the level of non-hemoglobin heme in reticulocytes, also inhibited [2–14C]-glycine into heme but not into protoporphyrin. With high concentrations of ferric pyridoxal benzoylhydrazone (Fe-PBH), which, independent of transferrin and transferrin receptors, can be used as a source of iron for heme synthesis in reticulocytes, significantly more iron is incorporated into heme than from saturating concentrations of Fe-transferrin. This suggests that some step (or steps) in the pathway of iron from extracellular transferrin to protoporphyrin limits the overall rate of heme synthesis in reticulocytes. In addition, hemin in concentrations that inhibit the utilization of transferrin-bound iron for heme synthesis has no effect on the incorporation of iron from Fe-PBH into heme. Our results indicate that in reticulocytes heme inhibits and controls the utilization of iron from transferrin but has no effect on the enzymes of porphyrin biosynthesis and ferrochelatase. This mode of regulation of heme synthesis may be a specific characteristic of the hemoglobin biosynthetic pathway.

Ferric pyridoxal isonicotinol hydrazone can provide iron for heme synthesis in reticulocytes

We have examined whether reticulocytes depleted of transferrin might incorporate 59Fe from 59Fe-labelled pyridoxan isonicotinoyl hydrazone (PIH). Transferrin-depleted reticulocytes showed a time-, temperature- and concentration-dependent incorporation of 59Fe when incubated with 20–200 μM 59Fe-PIH. The amount of 59Fe incorporated with 200 μM 59Fe-PIH is equal to or higher than that taken up from transferrin at 20 μM 59Fe concentration. After 60 min about 60% of the 59Fe taken up by the cells is recovered in heme while the remainder is probably still bound to PIH. 1 mM succinyl acetone (a specific inhibitor of heme synthesis) inhibits PIH-mediated incorporation of 59Fe into heme by about 79% indicating that 59Fe from 59Fe-PIH is incorporated into de novo synthesized protoporphyrin. As is the case with transferrin, erythrocytes do not incorporate 59Fe from 59Fe-PIH. Pretreatment of reticulocytes with pronase does not inhibit their ability to incorporate 59Fe from 59Fe-PIH, suggesting that, unlike the uptake of Fe from transferrin, membrane receptors are not involved in the uptake of Fe-PIH by the cells.

Additive inhibition of in vitro rabbit reticulocyte heme and protein synthesis with combinations of isoniazid, chloramphenicol and ethanol.

Isoniazid, chloramphenicol and ethanol are well known hematopoietic toxins. These agents inhibit in vitro rabbit reticulocyte heme synthesis and, as a result, inhibit protein synthesis. When combinations of isoniazid (2 x 10(-3)M) and ethanol (0.1M), isoniazid and chloramphenicol (10(-3)M) or ethanol and chloramphenicol were present in the same incubation, their inhibitory effects on heme and protein synthesis were additive. These results suggest that the development of clinical hematological toxicity in man may result from additive interaction of chemically unrelated compounds which inhibit the heme synthetic pathway.

Benzene and ethanol additive inhibition of rabbit reticulocyte heme and protein synthesis

Benzene and ethanol are two well-known hematopoietic toxins. Both of these agents inhibit reticulocyte heme synthesis, and as a result inhibit protein synthesis. When these two compounds are present in the same incubation, their effects are additive. These in vitro results suggest further evaluation of the combined toxicity of these two chemically unrelated agents is indicated. It is also suggested that it might be prudent for workers exposed to benzene to limit their ingestion of alcohol.

The reticulocyte-mediated release of iron and bicarbonate from transferrin: Effect of metabolic inhibitors

The effect of three groups of metabolic inhibitors on the incorporation of Fe and release of bicarbonate from transferrin by rabbit reticulocytes was measured. Inhibitors which affect reticulocyte Fe and transferrin uptake to the same extent (sodium arsenite, N-ethylmaleimide and iodoacetamide); those which inhibit reticulocyte Fe uptake to a greater extent than transferrin uptake (NaN 3, NaF, NaCN, rotenone, oligomycin, 2,4-dinitrophenol and cycloheximide); and compounds which after reticulocyte heme synthesis (CoCl 2, isonicotinic acid hydrazide and hemin) were used. In each case the effect on Fe incorporation and bicarbonate release was the same Thus, additional evidence has been obtained for the idea that the reticulocyte-mediated release of Fe and bicarbonate from transferrin are tightly coupled. The results are consistent with the hypothesis that an enzymatic attack on transferrin-bound bicarbonate is involved in the removal of Fe from transferrin by erythroid cells.

Ferrioxamine Iron in Rabbit Reticulocyte Heme Synthesis

Abstract Ferrioxamine B, a naturally occurring sideramine, serves as an iron transport agent and iron donor for heme synthesis in micro organisms. It is unable to effectively replace transferrin in iron delivery for rabbit reticulocyte hemoglobin formation.

Problems of reduced heme synthesis in reticulocytes with impaired globin synthesis.

The 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.