Background/aims: The erythrocyte is a cell exposed to a high level of oxygen pressure and to oxidative chemical agents. This stress involves SH-groups oxidation, cell shrinkage by activation of K-Cl cotransport (KCC) and elevation of the band 3 tyrosine phosphorylation level. The aim of our study was to test whether oxidative stress could influence band 3-mediated anion transport in human red blood cells. Methods: To evaluate this hypothesis, normal and pathological (glucose 6 phosphate dehydrogenase (G6PDH) defficient) erythrocytes were treated with known sulphydryl-blocking or thiol-oxidizing agents, such as N-ethylmaleimide (NEM), azodicarboxylic acid bis[dimethylamide] (diamide), orthovanadate, Mg 2+ and tested for sulphate (SO 4 -) uptake, K + efflux, G6PDH activity and glutathione (GSH) concentration. Results: In normal red blood cells, the rate constants of SO 4 -uptake decreased by about 28 % when cells were incubated with NEM, diamide and orthovanadate. In G6PDH-deficient red blood cells, in which oxidative stress occurs naturally, the rate constant of sulphate uptake was decreased by about 40% that of normal red cells. Addition of oxidizing and phosphatase inhibitor agents to pathological erythrocytes further decreased anion transport. In contrast, G6PDH activity was increased under oxidative stress in normal as well as in pathological cells and was lower in the presence of exogenous Mg 2+

Anion transport in the trout red blood cell is mediated by a membrane protein that selectively binds dihydro-4,4'-dithiocyanostilbene-2,2'-disulfonic acid (3H2DIDS) and that forms on sodium dodecyl sulfate (SDS)-polyacrylamide gel electropherograms a band with the same diffuse structure at the same location as the band 3 protein of the mammalian red blood cells. There exists a linear relationship between binding of H2DIDS to this protein and the inhibition of anion equilibrium exchange. At maximal inhibition about 8 X 10(6) molecules/cell are bound to the protein. The kinetics of anion transport in the trout red blood cell differ from those of mammalian red blood cells. In addition to a H2DIDS-sensitive component of sulfate transport there exists a considerable H2DIDS-insensitive component with a relative magnitude that decreases with increasing temperature. At 23 degrees C, it amounts to about 25%. The temperature dependence of the H2DIDS-sensitive component is about 15 kcal/mol instead of 32 as in human red blood cells. Cl- transport increases with increasing pH. Above pH 7.4, the rate of transport becomes too fast to be measurable with either inhibitor stop or filtration technique. SO2-4 transport is nearly pH independent over the pH range 6.5 to 7.8 and the net entry of SO2-4 in exchange against intracellular Cl-, as followed in the absence of CO2, is accompanied by little if any proton uptake. Net proton uptake becomes measurable only at temperatures above 40 degrees C. Possibly at lower and more physiological temperatures, the band 3 protein in the red blood cell of the trout accomplishes part of the SO2-4 movements without cotransporting protons.

Sulfate transport by band-3 protein in adult human erythrocytes was shown to be modulated by oxygen pressure. In particular, a higher transport activity was measured under high oxygen pressure than at low one (0.0242+/-0.0073 vs. 0.0074+/-0.0010 min(-1)). Other factors, such as magnesium ions and orthovanadate, which can indirectly affect the binding properties of the cytoplasmic domain of band 3 (cdb3), influence significantly the anion exchanger activity. No effect of oxygen pressure on sulfate transport was found in chicken erythrocytes, which may be related to their lacking the cdb3 binding site. These findings are fully consistent with a molecular mechanism where the oxygen-linked transition of hemoglobin (T-->R) could play a key role in the regulation of anion exchanger activity.

This study aimed to show that modifications in intracellular metabolism are implicated in the pathophysiology of diabetes mellitus and essential hypertension. In fact, total magnesium, calcium, sodium and potassium concentrations, measured in the erythrocytes of normotensive, diabetic and hypertensive patients, have given the following results: a lower intracellular potassium concentration in the erythrocytes of diabetic and hypertensive patients than the erythrocytes of normotensive patients and a more elevated sodium, magnesium, calcium concentrations in the erythrocytes of diabetic and hypertensive patients than the normotensive. Because of the importance of Mg2+ and Ca2+ in metabolic enzyme regulation and their interaction with both Hb and band 3 protein, we examined SO4(2-) kinetic influx in the erythrocytes of normotensive, hypertensive and diabetic patients. The kinetic plots showed different profiles over the three groups and the fluxes were found to be 0.024, 0.061 and 0.072 mmol x (l cells x min)(-1) in normotensive, hypertensive and diabetic patients, respectively. We also found that the Vmax and Km of sulphate influx, obtained by Hofstee plots, increased in the erythrocytes of hypertensive and diabetic patients compared with control cells. In contrast, sulphate influx in the erythrocytes of diabetic and hypertensive patients in the presence of Nifedipine, a calcium antagonist, showed no difference either in the rate constants or in the kinetic profiles, compared to the normotensive control subjects.

The anion influx was measured in order to study the interaction among organic phosphates, magnesium, haemoglobin and the N-terminal of the cytoplasmic domain of band 3 protein in human, chicken and trout erythrocytes. The rate constant for SO(4)(2-) influx in human and trout erythrocytes increased significantly when it was measured with an increased concentration of intracellular Mg(2+). The SO(4)(2-) influx was also measured in human erythrocyte ghosts in the presence and absence of Mg(2+). The smaller activation provoked by Mg(2+) in ghosts could be caused by the presence of a small quantity of haemoglobin which remained inside. The SO(4)(2-) uptake in chicken erythrocytes in the presence and in absence of Mg(2+) was characterized by very similar rate constants. The results suggest that the small increase in intracellular Mg(2+) in the erythrocytes involves an increase in the formation of Mg(2+)-ATP and Mg(2+)-2,3 BPG complexes reducing the affinity of the organic phosphates for Hb. This new situation may influence the functions of the anion transporter with consequent variations of SO(4)(2-) influx throughout the erythrocyte membrane in human and in trout erythrocytes, whereas in chicken RBCs this function cannot occur and, in fact, no increase in sulphate influx was noticeable. The measurement of Hb/O(2) affinity by the use of alternating fixed and variable concentrations of organic phosphates and Mg(2+), confirms the interactions between these elements and their effect on the mechanism of the affinity. When we measured the sulphate influx in the presence of DIDS we found some differences in the three types of cells.