cyclic-gmp and zileuton

Hydroxyurea (HU) induces fetal hemoglobin synthesis through activation of cyclic guanine monophosphate (cGMP) signaling. Studies in sickle cell patients demonstrated increased circulating nitric oxide (NO) levels after oral HU treatment. However, the direct measurement of NO in erythroid cells and its role in fetal hemoglobin induction have not been defined. Therefore, we quantified the level of nitrate and nitrite (NOx) generated by HU in human erythroid progenitors in the presence of three nitric oxide synthase inhibitors (NOS), including N(G)-monomethyl-L-arginine (L-NMMA). In addition, cGMP levels were measured in the presence or absence of the pathway inhibitor 1H-(1,2,4)ox-adiazolo(4,3-a)quinoxalin-1-one, which blocks soluble guanylyl cyclase formation. HU treatment increased NOx levels and gamma-globin transcription in K562 and primary erythroid cells, which was augmented when HU was combined with L-NMMA. Pretreatment with the cGMP pathway inhibitor reversed gamma-gene activation by HU. These data demonstrate the direct stimulation of cellular NO and cGMP signaling in erythroid progenitors by HU as a possible mechanism for gamma-globin gene activation.

Topics: Cyclic GMP; Erythroid Cells; Fetal Hemoglobin; gamma-Globins; Humans; Hydrogen Peroxide; Hydroxyurea; K562 Cells; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitrites; omega-N-Methylarginine; Signal Transduction; Stem Cells; Transcription, Genetic; Transcriptional Activation

Induction of fetal hemoglobin (Hb F) is an important therapeutic tool in ameliorating complications of sickle cell disease. Nitric oxide has been implicated in the mechanism of Hb F synthesis induced by hydroxyurea (HU). This study examined whether zileuton (ZL), a structural analog of hydroxyurea, possessed Hb F-inducing properties and the potential role nitric oxide plays. ZL caused a dose-dependent increase in gamma-globin expression in K562 cells. This effect was confirmed by a dose-dependent increase in Hb F synthesis in erythroid progenitors from individuals with sickle cell anemia and normal hemoglobin genotypes. l-arginine had no effect on Hb F production; however, it dose-dependently inhibited ZL's ability to induce Hb F. The nitric oxide synthase inhibitor N(G)-monomethyl-l-arginine (l-NMMA) inhibited l-arginine's effect and restored ZL-mediated increase in Hb F synthesis. In addition, 8-PCPT-cGMP (8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate) inhibited ZL-mediated induction of Hb F synthesis. When comparing l-NMMA effects alone on ZL and HU, a partial reversal of increased Hb F synthesis was seen only with HU. Neither l-arginine alone nor l-arginine in combination with l-NMMA effected hydroxyurea-mediated induction of Hb F synthesis. This study demonstrates that ZL induces Hb F through a mechanism that involves l-arginine/nitric oxide/cGMP in a manner distinctly different from HU.

Topics: Anemia, Sickle Cell; Arginine; Case-Control Studies; Cells, Cultured; Cyclic GMP; Dose-Response Relationship, Drug; Erythroid Precursor Cells; Fetal Hemoglobin; Gene Expression Regulation; Humans; Hydroxyurea; K562 Cells; Nitric Oxide; Nitric Oxide Synthase; Signal Transduction