Red blood cells (RBCs) from sickle cell patients, which are exposed to a greater oxidative challenge than normal, have elevated K⁺ transport. This characteristic is responsible for their dehydration. As part of a study to investigate the mechanisms responsible for this transport phenotype, we have examined the effects of oxidants on passive K⁺ transport in RBCs (Muzyamba et al. 2000, 2001). Stimulation of the K⁺ÐCl^– cotransporter (KCC) correlated with both reduction of reduced glutathione (GSH) and accumulation of methaemoglobin (metHb), not GSH alone, whilst loss of O₂ dependence of KCC was independent of GSH but correlated with accumulation of metHb. Here we report the effects of nitrite (NO₂^–), which stimulates KCC in RBCs from sheep and horse (Adragna & Lauf, 1998; Muzyamba et al. 2000), on human RBCs.

Normal human RBCs, taken with ethical permission from consenting volunteers, were pretreated for 60 min at 10 % haematocrit (Hct) with or without NO₂^– (1, 3 or 5 mM). Cells were then equilibrated in air or N₂ after which transporter activity (80 mM K⁺, 2.5 mM Ca²⁺) was measured at 4 % Hct using ⁸⁶Rb⁺ as a K⁺ congener, in the presence of ouabain (0.1 mM) and bumetanide (1 mM) to obviate transport through Na⁺/K⁺ pump and Na⁺ÐK⁺ÐCl^– cotransporter. KCC activity was determined as the Cl^– -dependent component of K⁺ influx (Cl^– replaced with nitrate). We also measured activity of the Ca²⁺-activated K⁺ channel (Gardos channel or I_K) as the clotrimazole (5 mM)-sensitive component.

Increasing [NO₂^–] produced progressive stimulation of KCC (3.5-fold at 1 mM; 6.5-fold at 5 mM) and loss of O₂ dependence (deoxygenation inhibited KCC in control cells by 90 % but only about 50 % in NO₂^–-treated cells) (Table 1; P < 0.05, Student’s paired t test). As for other oxidants, treatment with NO₂^– caused accumulation of metHb (> 60 % at 5 mM) and depletion of GSH. I_K, however, was not activated in either control cells or following treatment with NO₂^–.

In conclusion, NO₂^–, like other oxidants, has profound effects on membrane transport in human RBCs altering the response to O₂ tension. Elucidating the target of oxidants will be invaluable for understanding the transport abnormalities observed in sickle cells.

This work was supported by Action Research and The Wellcome Trust.

All procedures accord with current local guidelines and the Declaration of Helsinki.