Melatonin ameliorates anion exchange capability through Band 3 protein after H2O2-induced oxidative stress.

The activity of Band 3 protein (B3p), which mediates Cl−/HCO3− exchange through erythrocytes membrane, can be monitored by measuring the rate constant for SO4= uptake and is reduced by H2O2-induced oxidative stress. The aim of the present study was to prove whether melatonin, hormone produced by pineal gland during the dark phase of the cyrcadian cycle and known as antioxidant compound, though provoking lipid peroxidation on cell membranes at certain doses, may ameliorate B3p efficiency when oxidative stress is applied. As a first step, MDA assay was performed on human erythrocytes (10% hematocrit) treated with melatonin (1-500 µM) to exclude lipid peroxidation. Successively, erythrocytes (3% hematocrit) pre-treated (1 h, 37 °C) with 100 µM melatonin, were submitted to H2O2 (300 µM, 30 min at 25 °C), chosen to model oxidative stress conditions, and the rate constant for SO4= uptake was determined by a turbidimetric method. Melatonin alone, at a concentration not inducing lipid peroxidation, does not affect erythrocyte morphology and, when applied prior to H2O2 treatment, prevents cell shrinkage due to oxidative stress and restores the rate constant for SO4= uptake. Hence, we may suggest that: i) oxidative stress induced by 300 µM H2O2, comprised in a physiological range, affects B3p function by reducing anion exchange capability with no lipid peroxidative event; ii) such effect is reversed by melatonin pre-treatment; iii) melatonin impairs the reduction in B3p expression levels caused by H2O2; iv) an antioxidant effect of melatonin on B3p can be suggested; v) monitoring of B3p anion exchange capability under oxidative stress is a useful tool to check erythrocytes homeostasis and determine the beneficial effects of natural antioxidants.