Oxidant-mediated modification of the cellular thiols is sufficient for arginase activation in cultured cells.

Increased arginase activity in the vasculature has been implicated in the regulation of nitric oxide (NO) homeostasis, leading to the development of vascular disease and the promotion of tumor cell growth. Recently, we showed that cysteine, in the presence of iron, promotes arginase activity by driving the Fenton reaction. In the present report, we showed that induction of oxidative stress in erythroleukemic cells with the thiol-specific oxidant, diamide, led to an increase in arginase activity by 42% (P = 0.02; vs. control). By using specific antibodies, it was demonstrated that this increase correlated with an increase in arginase-1 levels in the cells and with corresponding decreases in glutathione and protein thiol levels. Treatment of cells with aurothiomalate (ATM), a protein thiol-complexing agent, diminished the activity of arginase and arginase-1 levels by 19.5 and 35.2%, respectively (vs. control) and significantly decreased both glutathione and protein thiol levels, further implicating the thiol redox system in the cellular activation of arginase. Furthermore, diamide significantly altered the kinetics of arginase, resulting in the doubling of its V(max) (vs. control). Our presented data demonstrate, for the first time that the intracellular arginase activation is may be enhanced in part, via a cellular thiol-mediated mechanism.

The redox state of the glutathione/glutathione disulfide couple mediates intracellular arginase activation in HCT-116 colon cancer cells.

BACKGROUND: Emerging studies have implicated arginase hyperactivity in the dysregulation of nitric oxide synthesis, which can lead to the development of vascular disease and the promotion of tumor cell growth. Recently, we showed that cysteine, in the presence of molecular iron, promotes arginase activity by driving the Fenton reaction. However, the exact mechanism of arginase activation in the cell induced by oxidative stress is unknown. AIM: The aim of the present study is to examine whether intracellular arginase is regulated by the cellular redox status of glutathione. METHOD: To test this hypothesis, the glutathione/glutathione disulfide redox couple was altered in colon cancer cells with the thiol-specific oxidant, diamide, or the glutathione inhibitor, buthionine-(S,R)-sulfoximine, and the activity of the arginase in the cells was assessed. RESULTS: Treatment of cells with diamide, a thiol-specific oxidant, resulted in a dose-dependent decrease in the glutathione/glutathione disulfide ratio that was associated with the loss of glutathione and a coincident increase in arginase activity and arginase-1 levels in drug-treated cells compared with untreated cells. These results show that oxidation-induced redox changes of glutathione are of sufficient magnitude to control the activity of arginase in the cells. Thus, the physiologic modulation of the glutathione/glutathione disulfide ratio could prove to be a fundamental parameter for the control of arginase activity in pathological conditions of increased oxidative stress. CONCLUSION: This is the first evidence supporting the ex vivo regulation of arginase activity through the redox modulation of intracellular glutathione. The potential adaptive and pathological consequences of glutathione redox regulation of arginase activity are discussed.

Cysteine-iron promotes arginase activity by driving the Fenton reaction.

Impairment of nitric oxide bioavailability secondary to increased arginase activity and overproduction of reactive oxygen species (ROS) is thought to be a major cause of vascular complications in sickle cell disease (SCD). However, the role of ROS in the induction of arginase activity is unknown. This study investigated whether the mechanism of arginase activation involves the ROS produced during oxidative stress. Our study reveals that cysteine-iron dose-dependently stimulated arginase activity with a corresponding increase in (.)OH radical formation. The ()OH radicals produced were significantly inhibited by salicylic acid derivatives and superoxide dismutase. Surprisingly, the inhibition of (.)OH radicals parallels the inhibition of arginase activity, thus suggesting the role of cysteine-iron in the stimulation of arginase via the Fenton reaction. This is the first evidence demonstrating the participation of (.)OH radicals in the stimulation of arginase activity, and thus provides novel avenues for therapeutic modalities in hemoglobinopathies and other inflammation-mediated diseases.

In vitro evidence of the inhibitory capacity of chloroquine on arginase activity in sickle erythrocytes.

Increased levels of erythrocyte arginase activity are believed to play a key role in the pathogenesis of sickle cell disease (SCD). Because increased arginase activity has been implicated in the exacerbation of pulmonary hypertension in SCD, this enzyme is considered an important therapeutic target for identifying new drugs to treat and manage SCD and other inflammatory disorders. Although chloroquine (CQ) is prescribed as an anti-malarial and anti-rheumatoid drug, the mechanism of its anti-inflammatory activity is largely unknown. The present study found that CQ inhibited arginase in a dose-dependent manner, and also displayed a linear competitive inhibition on sickle erythrocyte arginase. The apparent K(M) values in the presence of inhibitor were considerably reduced at both physiological and slightly acidic pHs. Slope replots of the double reciprocal plots at pH 6.8 and 7.4 also indicated simple competitive inhibitory mechanism of CQ, and Ki values for CQ were within micromolar levels. To our knowledge, this is the first example of an anti-malarial and anti-inflammatory agent displaying competitive inhibition kinetics on arginase. The outcome of this study may provide a template for the rational design of specific agents either alone or in combination with CQ for the inhibition of arginase activity.

Modulation of erythrocyte arginase activity in sickle cell disease patients during hydroxyurea therapy.

An elevated erythrocyte arginase activity with a corresponding decrease in nitric oxide (NO) level has been implicated in the pathophysiology of sickle cell disease (SCD). Recent studies have shown that hydroxyurea (HU) increases the production of NO, which increases the soluble guanylate cyclase activity and fetal haemoglobin (HbF) synthesis. To study the effects of HU on the arginase and nitric oxide synthase (NOS) activities in SCD patients, we compared levels of arginase activity and NO metabolites in red blood cells and plasma, respectively, from 23 patients with SCD (HbSS) receiving HU therapy, with those of 12 SCD patients not receiving HU treatment. Patients on HU therapy showed significantly lower arginase activity than that of HbSS patients not on HU therapy (1.36+/-0.2 U/10(8) cells vs. 3.31+/-0.29 U/10(8) cells). NOS activity was higher in patients on HU therapy than in untreated patients (0.72+/-0.4 nmol/ml/min vs. 0.35+/-0.15 nmol/ml/min, P<0.05). Among the HU-treated patients, the decreased level of arginase activity correlated (r=0.71) with HbF level as well as the mean corpuscular haemoglobin content. These data suggest that one of the beneficial effects of HU in vivo may involve the regulation of arginase activity and a concomitant induction of NOS activity, which may lead to an increased production of NO. The outcome of this study may lead to the development of improved NO-based treatments for SCD.

Niprisan (Nix-0699) improves the survival rates of transgenic sickle cell mice under acute severe hypoxic conditions.

The substitution of glutamic acid by valine at the sixth position of the beta-globins of haemoglobin S (Hb S) causes a drastic reduction in the solubility of the deoxy form of Hb S. Under hypoxic conditions, deoxy-Hb S molecules polymerize inside the cells, forming rigid, sickled cells. We studied the effect of Niprisan (Nix-0699), a naturally occurring antisickling agent, on the survival of transgenic (Tg) sickle mice under severe acute hypoxic conditions (60 min). Before hypoxia exposure, the mice were treated by gavage once daily for 7 d with 0 mg/kg (n = 10), 10 mg/kg (n = 5), 50 mg/kg (n = 5), 300 mg/kg (n = 4) or 500 mg/kg (n = 5) of Nix-0699. The mean survival times of the untreated and treated mice were 10, 25, 39, 55 or 60 min respectively. The percentage of sickled cells in the venous blood of the treated mice was lower than that in control mice and was dose dependent. Histological examination of the lungs of the control mice showed entrapment of massive numbers of sickled cells in the alveolar capillaries, although the degree of such entrapment decreased with the increased dose of Nix-0699. Nix-0699 may be a promising option for the treatment and management of patients with sickle cell disease.

Combined use of nonmyelosuppressive nitrosourea analogues with hydroxyurea in the induction of F-cell production in a human erythroleukemic cell line.

OBJECTIVE: Although hydroxyurea (HU) has been used clinically to treat patients with sickle cell disease (SCD), not all patients benefit from HU treatment due to its toxicity. The objective of this study was to investigate the effectiveness of the use of two new Hb F-inducing nitrosourea analogues, 2-[3-(2-methyl, 2-nitroso) ureido]-2-deoxy-D-glucopyranose (MNGU) and 2-[3-(2-chloroethyl) ureido]-2-deoxy-D-glucopyranose (CGU), in combination with HU in K562 cells or erythroid progenitors. MATERIALS AND METHODS: After K562 cells were cultured with different concentrations of HU with CGU or MNGU, aliquots of the cells were obtained to determine the total (benzidine-positive) hemoglobin level, number of F cells, and Hb F level. Erythroid progenitor cells of SCD patients and healthy donors were cultured with the optimal drug concentrations, and the number of BFU-E and Hb F level were determined. RESULTS: Our results showed that the combined use of HU with CGU or MNGU increased the number of both benzidine-positive normoblasts and F cells in a synergistic manner. Further, a lower concentration of HU was required to induce a significant level of Hb F synthesis when combined with either of the two compounds in comparison with treatment with HU alone. On day 4, the number of benzidine-positive cells was 4.5- to 6.5-fold and the number of F cells was 5.0- to 8.0-fold higher than the respective numbers in the untreated K562 cells. Similarly, a 3.2- to 14.3-fold induction of Hb F was obtained when human erythroid progenitors from SCD patients were treated with the same drug combinations. CONCLUSION: Based on these results, the use of CGU or MNGU in combination with HU might offer substantial benefits to patients with SCD and other hemoglobinopathies.

In vitro effects of NIPRISAN (Nix-0699): a naturally occurring, potent antisickling agent.

Among the various potential antisickling agents tested, hydroxyurea (HU) has been the most effective compound used for the treatment of patients with sickle cell disease (SCD). Although HU is effective in many patients, not all patients respond to this drug. In addition, some patients reveal adverse effects, including myelosuppression. In an attempt to find other effective agents with less adverse effects, we investigated the antisickling effect of NIPRISAN (Nix-0699). We found that Nix-0699, an ethanol/water extract from indigenous plants, has a strong antisickling effect. The concentration of Nix-0699 required to inhibit 50% of erythrocyte sickling was about 0.05 mg/ml. As for the kinetics of polymerization, addition of 0.05 microg/ml Nix-0699 caused a sixfold prolongation of the delay time prior to deoxy-Hb S polymerization when compared with that of untreated Hb S samples. The solubility of deoxy-Hb S significantly increased upon treatment with Nix-0699. Analysis of the effect of Nix-0699 on the Hb S oxygen affinity indicated that the drug slightly shifted the oxygen-dissociation curve of Hb S toward the left without any apparent change in the Hill coefficient. These results suggest that the antisickling properties of Nix-0699 may involve direct interaction with Hb molecules. Incubation of red blood cell (RBC) suspensions with various concentrations of Nix-0699 did not dehydrate RBCs, cause haemolysis, increase the amount of denatured Hb, nor form met-Hb. In view of the outcome of this study, Nix-0699 may be a promising option for the treatment of patients with SCD.