losartan-potassium and succinylacetone

Erythropoietin (Epo) is one of the physiologically important genes whose transcription is up-regulated by hypoxia. Our laboratory previously proposed that the sensor of this event is a heme protein which turns over rapidly. We have investigated the effects of four inhibitors of heme synthesis (4,6-dioxoheptanoic acid (DHA), isoniazid (INH), N-methyl protoporphyrin IX (MPP), and deferoxamine mesylate (DSF)) on hypoxia-, cobalt-, and DSF-induced erythropoietin (Epo) mRNA expression, heme biosynthesis, and cell viability in Hep3B cells. DHA (0.1-1.0 mM) inhibited heme biosynthesis more than 85%, but did not suppress Epo mRNA expression. Epo mRNA expression was inhibited only at higher concentrations of DHA (2, 4 mM) which also inhibited cell viability. No suppression of Epo mRNA expression by INH was observed at doses known to inhibit heme biosynthesis. MPP did not suppress Epo mRNA expression although it showed an inhibitory effect on heme biosynthesis without any decreased cell viability. 130 microM DSF, a dose which inhibited heme biosynthesis without cell toxicity, suppressed hypoxia-induced Epo mRNA expression, but enhanced cobalt-induced Epo mRNA expression. These results show that although the oxygen sensor is probably a heme protein it does not turn over rapidly. Therefore, cobalt is unlikely to act by substituting for heme iron.

Topics: Cell Survival; Deferoxamine; Enzyme Inhibitors; Erythropoietin; Heme; Heptanoates; Humans; Isoniazid; Protoporphyrins; Tumor Cells, Cultured

Heme synthesis by erythroid progenitor cells is maintained by erythropoietin (EP), insulin-like growth factor-I (IGF-I), and stem cell factor (SCF), and without these growth factors apoptosis (programmed cell death) occurs. To clarify the possible interaction between heme synthesis and programmed cell death of human erythroid progenitor cells, the effect of specific inhibition of heme synthesis on apoptosis of highly purified human erythroid colony forming cells (ECFC) was studied. When the amount of uncleaved DNA was determined as a measure of apoptosis, the heme synthesis inhibitors, succinylacetone (SA) (0.1 mmol/L) or isonicotinic acid hydrazide (INH) (10 mmol/L), significantly decreased the amount of uncleaved DNA (P < 0.01) in the presence of erythropoietin (EP). Addition of recombinant heavy-chain ferritin (rHF) (10 nmol/L), or deprivation of transferrin from the culture medium, which decreased heme synthesis, also reduced the amount of uncleaved DNA (P < 0.01). The production of apoptosis by diverse inhibitors of heme synthesis was in each case reversed by the addition of hemin (0.1 mmol/L) and did not occur with HL-60 cells. When the colony-forming capacity of ECFC was determined by plasma clot assay, SA, INH, or rHF reduced the number of CFU-E (P < 0.01), and the effect of SA was reversed by hemin. The addition of SA did not alter the c-myc response of ECFC to EP. These data indicate that inhibition of heme synthesis induces apoptosis of human erythroid progenitor cells, in a manner independent of an early c-myc response, and suggest that the presence of apoptosis in ineffective erythropoiesis may be secondary to impaired heme synthesis.

Topics: Apoptosis; Cells, Cultured; Culture Media; Erythroid Precursor Cells; Erythropoietin; Ferritins; Heme; Heptanoates; Humans; Isoniazid; Proto-Oncogene Proteins c-myc; Recombinant Proteins; Stem Cells; Transferrin

Erythropoietin (Epo) was found to act as a concentration-dependent inducer of aminolevulinic acid (ALA) synthase and porphobilinogen (PBG) deaminase in normal human bone marrow in culture. Epo increased enzymatic activities in individual plated nucleated cells. At a low concentration of Epo, heme oxygenase activity did not change in human bone marrow erythroid progenitor cells. However, Epo at a concentration of 2 U/ml increased heme oxygenase as demonstrated by an increase in both the enzyme protein and its mRNA. In experiments with an inhibitor of heme synthesis, succinylacetone (SA), Epo failed to stimulate erythroid colony-forming unit (CFU-E) growth, but this CFU-E inhibition by SA was completely overcome by the addition of hemin. Epo nevertheless potentiated induction of ALA synthase in the presence of SA. Hemin exerted its regulatory role by negative feedback on ALA synthase in the presence of SA and Epo. Heme potentiated Epo action and resulted in the increase of human marrow erythroid progenitor cell proliferation and differentiation and a concomitant stimulation of ALA synthase and PBG deaminase. The potentiating effects of hemin on CFU-E growth were observed in human bone marrow cells cultured in media supplemented with fetal calf serum or serum-free media with interleukin 3 (IL-3). These results indicate that Epo is a potent inducer of ALA synthase and PBG deaminase in normal human bone marrow. In addition, our results may explain the mechanisms by which heme potentiates Epo or IL-3 enhancement of erythropoiesis. 1) Heme may stimulate the translation of several globin and nonglobin mRNAs, including those of ALA synthase and PBG deaminase; 2) as endogenous cellular heme synthesis reaches optimal levels, heme exerts its regulatory role on ALA synthase by negative feedback inhibition. Additionally, an increase in cellular heme may lead to an increase in its own degradation by induction of heme oxygenase.

Topics: 5-Aminolevulinate Synthetase; Bone Marrow; Bone Marrow Cells; Cells, Cultured; Drug Synergism; Erythrocytes; Erythropoietin; Hematopoietic Stem Cells; Heme; Heme Oxygenase (Decyclizing); Hemin; Heptanoates; Humans; Hydroxymethylbilane Synthase; Osmolar Concentration; Reference Values

The effects of 4,6-dioxoheptanoic acid (succinylacetone, SA), an inhibitor of delta-aminolevulinic acid dehydratase, on total iron uptake, heme synthesis, and globin synthesis were studied in rat marrow cells in culture in order to examine the coordination of heme and globin synthesis. SA inhibited heme synthesis in both control and erythropoietin-stimulated cells in a dose-dependent fashion; at 10(-3) M, inhibition was complete, whereas at 10(-7) M, there was no significant effect. Inhibition of total iron uptake was also dose-dependent although, at 10(-3) M, it was not complete. The inhibition of heme synthesis by SA was partially overcome by addition of 10(-4) M porphobilinogen or protoporphyrin IX. SA caused an almost complete suppression of globin formation in both erythropoietin-stimulated and unstimulated cells as early as five hours after the addition of the inhibitor. When inhibition of heme synthesis was incomplete, globin synthesis was partially inhibited. These results indicate that heme synthesis is required for erythropoietin-mediated induction of globin synthesis in cultured bone marrow cells.

Topics: Animals; Biological Transport; Bone Marrow; Cells, Cultured; Erythropoietin; Globins; Heme; Heptanoates; Iron; Male; Protein Biosynthesis; Rats; RNA