Hydroxyurea induces the eNOS-cGMP pathway in endothelial cells

Abstract

Hydroxyurea is a cell-cycle-specific drug that has been used to treat myeloproliferative diseases and sickle cell anemia. We have recently shown that hydroxyurea, like nitric oxide (NO)-donor compounds, increased cGMP levels in human erythroid cells. We show now that hydroxyurea increases endothelial-cell production of NO; this induction of NO in human umbilical vein endothelial cells (HUVECs) and human bone marrow endothelial cell line (TrHBMEC) is blocked by competitive inhibitors of NO synthase (NOS), such as N G -nitro-L-arginine-methyl ester (L-NAME) and N G -nitro-L-arginine. It is dependent on cAMP-dependent protein kinase (PKA) and protein kinase B (PKB/Akt) activity. We found that hydroxyurea dose-and time-dependently induced rapid and transient phosphorylation of eNOS at Ser1177 in a PKA-dependent manner; inhibitors of PKB/Akt could partially abrogate this effect. In addition, hydroxyurea induced cAMP and cGMP levels in a dose-dependent manner, as well as levels of intracellular calcium in HUVECs. These studies established an additional mechanism by which rapid and sustained effects of hydroxyurea may affect cellular NO levels and perhaps enhance the effect of NO in myeloproliferative diseases. IntroductionHydroxyurea is a cytostatic agent (S-phase inhibitor) that has been used to treat erythrocytosis and thrombocytosis in polycythemia vera and other myeloproliferative diseases. 1 More recently, hydroxyurea demonstrated therapeutic benefit for sickle cell anemia by increasing fetal hemoglobin (HbF). 2 In addition to the known inhibition of ribonucleotide reductase, hydroxyurea can be oxidized by heme groups to produce nitric oxide (NO) in vivo (in rats) and in humans. 3,4 Hydroxyurea reacts with oxyHb and deoxyHb to form metHb, which then reacts with another hydroxyurea to form iron nitrosyl hemoglobin (HbNO). 5 The formation of HbNO involves the specific transfer of NO from the -NHOH group of hydroxyurea by a series of intermediate reactions. 3 Additionally, in vivo production of NO may result from the hydrolysis of hydroxyurea to hydroxylamine, followed by the rapid reactions of hydroxylamine with Hb. 6 It has been reported that hydroxyurea administration significantly increased NO x (NO metabolites nitrite and nitrate) and cGMP levels in plasma of sickle cell patients. 7 We have recently shown that hydroxyurea, like NO-donor compounds, increased cGMP levels contributing to gamma-globin gene expression in human erythroid cells. 8 Hydroxyurea is completely absorbed and elimination is through both renal and nonrenal mechanisms. 9 The metabolism of hydroxyurea to NO most likely occurs in the liver. 3 Chemical oxidation of hydroxyurea with a variety of oxidants, including copper(II) ions, produces NO. 9,10 Hydroxyurea-treated vascular endothelial cells showed significant morphologic changes such as an increase in apparent cell size, accompanied by an increase in cell Na and K contents. 11 During hydroxyurea treatment, cells accumulate in the late G1 to early S phase of the cell cycle. 12 Also, the level...

Multiple Roles of Protein Kinase A in Arachidonic Acid–Mediated Ca2+ Entry and Tumor-Derived Human Endothelial Cell Migration

“…8): (a) phosphorylation and activation of eNOS and (b) direct and/or indirect phosphorylation of AA-activated channels. The first hypothesis, discussed previously, is supported by several reports on PKA-dependent NOS phosphorylation and activation (21,32,33). The second explanation, pointing to an effect on calcium channels, would be in agreement with PKAdependent phosphorylation of calcium-permeable channels reported for ECs and other cell types (34)(35)(36).…”

Relationship of erythropoietin, fetal hemoglobin, and hydroxyurea treatment to tricuspid regurgitation velocity in children with sickle cell disease

“…12 Hydroxyurea treatment lowers hemolysis 7,8 and decreases morbidity and mortality in patients with sickle cell disease. 33,34 Hydroxyurea may also have an impact on nitric oxide signaling by evoking nitric oxide synthase and decreasing arginine levels 9,35 ; the agent promotes the synthesis of nitric oxide by endothelial cells. 9 These factors may serve to protect from pulmonary hypertension.…”

“…1 Furthermore, once hemoglobin SC disease patients develop pulmonary hypertension, their prognosis is as poor as in hemoglobin SS patients with this complication. 6 Hydroxyurea decreases hemolysis 7,8 and induces nitric oxide in endothelial cells, 9 but the largest prospective studies of patients with sickle cell disease have not found less pulmonary hypertension among those receiving hydroxyurea. 1,3,10 It is also not clear whether high hemoglobin F levels reduce pulmonary hypertension risk in sickle cell patients.…”

Critical Reviews: How we treat sickle cell patients with leg ulcers

“…Several other authors are skeptical of a link between hydroxyurea and ulcers in SCD patients based on their own data [73][74][75], and many practical considerations preclude the randomized prospective trials needed for definitive evidence regarding this issue. There are many potential benefits of hydroxyurea use in SCD patients with ulcers: decrease in leukocyte count, decrease in inflammatory cytokines [76,77], increase in hemoglobin, increase in HbF, decrease in hemolysis, increase in oxygen carrying capacity, improved RBC rheology [78], and NO donor properties [79,80]. Our approach to the use of hydroxyurea in SCD is to aim for maximal fetal hemoglobin response, continue therapy unless there strong suspicion that hydroxyurea contributed to ulcer formation, and then consider switching to chronic transfusion therapy.…”