Increased iron absorption associated with intensive erythropoiesis and iron deposition as a result of continuous hemolysis contribute to cellular injuries in sickle cell disease (SCD). Iron overload seems to be a predisposing factor of disease severity and mortality in significant number of adult SCD and β-thalassemia patients. Indeed, there is now an impressive body of evidence indicating that increased intracellular iron pool might be the hallmark of mammalian cell susceptibility to oxidative stress. Recently, we showed that cysteine, in the presence of molecular iron, promotes arginase activity by driving the Fenton reaction in sickle erythrocytes (unpublished report). It has been suggested that the reduction of arginine bioavailability for nitric oxide (NO) production secondary to increased plasma arginase levels in SCD may contribute to severe pathophysiological derangements and increase mortality. We have previously shown that chloroquine (CQ), an anti-malarial and anti-rheumatoid drug, displays a linear competitive mode of inhibition on sickle erythrocyte arginase. In the present report we showed that treatment of cultured human erythroleukemic cells with 100 or 200 μM iron (ferric ammonium citrate) induced a significant increase in cell proliferation with corresponding increase in arginase activity. By using specific antibodies it was demonstrated that the increased arginase activity correlated with an increase in arginase-1 levels in the cells. Upon treatment of cells with 10 μM CQ, in the presence or absence of ferric ammonium citrate (FAC), the levels of arginase enzyme decreased by 33.5 % and 36.7 % respectively. In an effort to expand our understanding of the link between intracellular iron and CQ-mediated modulation of arginase activity, FAC-treated cells were co-incubated with 10 μM CQ for 48 hrs and the intracellular and membrane associated iron levels were determined by nitrilotriacetic acid (NTA)-ultracentrifugation assay. Our results indicate that CQ maximally enhanced the sequestration of intracellular iron by 39.3 % and that no effects of CQ were noted on the membrane associated iron levels. Further the level of ferritin was reduced by 29.4 % in CQ-treated cells in comparison with the FAC-treated cells alone as determined by immunosorbent assay. This data suggests that the observed diminished ferritin levels in the presence of CQ may be due to repression by translational regulators of ferritin function secondary to low intracellular iron levels, as evidenced in this study. Because treatment of cells with an intracellular iron chelator deferoxamine, in combination with CQ, caused a significant decrease in the levels of ferritin (60 % vs control; p = 0.006) and consequent reduction in ARG-1 levels (58.3 % vs control), we conclude that the sequestration of intracellular iron contributes in part to the CQ-mediated inhibition of arginase activity. We further investigated the effects of CQ on ferritin-Fe in a cell free system, reasoning that the effects of CQ on the intracellular iron pool may involve the modulation of iron mobilization from iron sources, such as ferritin (the main intracellular iron storage protein). To this end, purified human ferritin was incubated at predetermined time intervals in the presence or absence of 10–100 μM CQ at 37°C and the extent of NTA-mediated iron mobilization from ferritin was determined. Our results showed that CQ limits the mobilization of iron from ferritin in a dose-dependent manner. Our presented data demonstrate, for the first time, that CQ-mediated limitation of intracellular mobile iron pool modulates arginase activity as well as arginase-1 levels. We therefore speculate that an extreme increase in intracellular iron pool may result in the stimulation of arginase activity as observed in hemolytic diseases. In conclusion, we believe that our results represent a new and important improvement in understanding the pleiotropic effects of CQ on arginase activity, with specific reference to its participation on the limitation of intracellular iron availability in erythroid cells. Hence incorporating this template into structure-based enzyme design studies could lead to the production of new forms of efficient arginase inhibitors, which could be useful as therapeutic regimen in hemoglobinopathies and other iron overload related diseases.

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