Fig. 1.

Effect of WNK463 on KCl cotransport (KCC) activity in red cells from normal individuals (HbAA) and patients with sickle cell anaemia (SCA). Red cells from patients homozygous for SCA (20% haematocrit, Hct) or healthy individuals (40% Hct) were pre-incubated in N-MBS for 30 min at 37 °C in air in the presence of 0–40 nM WNK463, unless stated otherwise. They were then equilibrated in Eschweiler tonometers for 20 min in air (150 mmHg O₂) in the continued presence of WNK463, after which aliquots were diluted tenfold into flux tubes. KCC activity was measured as Cl^--dependent K⁺-influx for 10 min at an extracellular [K⁺] of 7.5 mM. KCC activity is given in mmol.(l cells.h)^-1. Ouabain (100 μM) and bumetanide (10 μM) were present in all experiments. a Effect of 0–40 nM WNK463 on KCC activity. KCC activity was normalised to that at 40 nM WNK463 and EC₅₀ calculated using nonlinear regression. b Effect of duration of pre-incubation with 40 nM WNK463 on KCC activity in HbSS cells. Symbols represent means ± SEM, n = 3. * p < 0.05, ** p < 0.01 compared to red cells incubated in the absence of WNK463