OXIDATIVE ACTIVATION OF K-CL COTRANSPORT BY DIAMIDE IN ERYTHROCYTES FROM HUMANS WITH RED-CELL DISORDERS, AND FROM SEVERAL OTHER MAMMALIAN-SPECIES

Red blood cells (RBCs) from different mammalian species were investiga ted for the presence of diamide-induced oxidative activation of K-Cl c otransport reported to be present in sheep but absent in human RBCs. K efflux was measured in RBCs from human with hemoglobin (Hb) A or S, g lucose-phosphate dehydrogenase (G6PDH) and a cytoskeletal deficiency, and from rat, mouse and rabbit. RBCs were incubated with diamide (0-1. 0 mM) in K-free Cl or NO3 media of variable osmolalities (200-450 mOsM ). CI-dependent K efflux or K-CI cotransport (estimated as the differe nce between K efflux rate constants in Cl and NO3) was activated by di amide in a sigmoidal fashion. Relative maximum K-CI cotransport follow ed the sequence: human HbA (1) < rabbit (1.8) < sheep (6.9) < human Hb S (9.5) similar to rat (9.7). Relative diamide concentrations for half maximal activation of K-CI cotransport followed the sequence: sheep ( 1.9) > human Hb A (1) > rabbit (0.75) > human HbS and rat (0.67). Cell swelling in 200 mOsM doubled K-CI cotransport in diamide, both in hum an HbA and S cells but reduced that in rat RBCs. In contrast, cell shr inkage at 450 mOsM obliterated K-CI cotransport in human HbA and S but not in rat RBCs. Human RBCs with G6PDH and a cytoskeleton deficiency behaved like HbA RBCs. In mouse RBCs, diamide-activated K-CI cotranspo rt was 30% higher in isotonic than in hypotonic medium. In human HbA a nd S, and in low or high K sheep RBCs fractionated by Percoll density gradient, diamide increased the activity of K-Cl cotransport, an effec t inversely correlated with cell density. Analysis of pooled data reve als that K-CI cotransport accounted for about 80% of all K flux in Cl. There was a statistically significant correlation between K-Cl cotran sport and K efflux in CI (P < 0.00001) and in NO3 (P < 0.00001). In co nclusion, a diamide-activated K-Cl cotransport was present in human RB Cs and in all other mammalian RBCs tested, with a large inter-, and fo r human and sheep, intraspecies variability for its maximum activity.