Effect of H2O2-induced oxidative stress on SO4= uptake through Band 3 protein.

Introduction. The erythrocyte membrane consists of a phospholipid bilayer with integral proteins associated to cytoskeleton through a proteins network. One of the most studied integral membrane proteins is Band 3 protein, particularly abundant in erythrocytes and responsible for Cl-/HCO3- as well as for cell membrane deformability, due to its cross link with cytoskeletal proteins. The membrane, as frequently exposed to oxidative events, represents a model to study the effect of oxidative stress. In an attempt to better clarify the response of Band 3 protein to external stressors, in the present study the effect of H2O2-induced oxidative stress on intact human erythrocytes and on hemoglobin-free resealed ghosts of erythrocytes has been considered. As SO4= uptake is mediated by Band 3 protein, the efficiency of this anion exchanger has been verified by measuring the rate constant for SO4= transport which is slower and better controllable than Cl- or HCO3- uptake and, hence, more easily estimated. Materials and methods. Oxidative conditions were induced by 30 min exposure of human erythrocytes (3% hematocrit) to different H2O2 concentrations (10 to 300 µM), with or without GSH (glutathione, 2 mM) or curcumin (10 µM), compounds with proved antioxidant properties. The rate constant for SO4= transport, while MDA (malondialdehyde) levels and –SH groups were estimated to quantify the effect of oxidative stress. Results. H2O2 induced a significant decrease in rate constant for SO4= uptake at both 100 and 300 µM H2O2. This reduction, observed in erythrocytes but not in resealed ghosts and associated to increase in neither MDA levels nor in –SH groups, was impaired by both curcumin and GSH, whereas only curcumin effectively restored H2O2-induced changes in erythrocytes shape. Conclusions. Band 3 protein function, monitored by SO4= uptake measurement, is affected by low concentrated H2O2, adding more information about the effect of oxidative stress at structural level, as Band 3 protein plays a role in cell membrane deformability due to its cross link with intracellular proteins and, in turn, in erythrocytes function as well as at functional level, namely correlated to efficiency of gas exchange. Our results show that: i) 30 min exposure to 300 µM H2O2 reduced SO4= uptake in human erythrocytes; ii) the oxidative damage was revealed by the reduction in rate constant for SO4= uptake, but not by changes in MDA or –SH groups levels; iii) the damage was produced via cytoplasmic components which cross link with Band 3 protein; iv) the natural antioxidant curcumin may be useful in protecting erythrocytes from oxidative injury, while GSH, commonly used in several experimental protocols, seems to be less effective in restoring SO4= uptake; v) SO4= uptake through Band 3 protein may be reasonably suggested as a tool to monitor erythrocytes function under oxidative conditions. Further investigations are needed to evaluate the impact of different conditions associated to increased reactive oxygen species (ROS) levels (aging, strenuous exercise, drug therapies and neurodegenerative diseases) on erythrocytes function.