Effect of urea on Rb influx in SS and AA RBCs. Fluxes were measured in unfractionated blood samples. (A) Rb influx versus urea concentration. Ouabain- and bumetanide-resistant (OBR) Rb influx was measured in HBS pH 7.4 in fresh RBCs (not swollen with nystatin) at various added urea concentrations and normalized to concurrently measured maximal volume-sensitive Rb influx in cells swollen to MCHC 270 g/L (27g/dL) or less by nystatin treatment. In Cl-free media, increasing urea concentration had no effect on OBR Rb influx (not shown). Symbols represent mean with error bars depicting SEM of 6 experiments. Curves were drawn by eye. The dashed line indicates 50% activation of KCC. (B) Rb influx versus urea concentration in swollen RBCs. Cells were swollen to initial MCHC 270 g/L (27 g/dL), and Rb influx was measured at various urea concentrations. Data are expressed as percentage of maximal volume stimulated flux ([urea] = 0). The 2 experiments shown with AA (open symbols) and SS (filled symbols) RBCs were independent of those in panel A. Lines were drawn by eye. (C) Rb influx versus MCHC in SS RBCs. Cells were swollen to various (initial) MCHC via nystatin treatment, and Rb influx was measured with 600 mM urea (▴) or without urea (▵). Maximal volume-stimulated flux was measured concurrently in cells with initial MCHC less than 270 g/L (27 g/dL) (without urea). Data are from 2 independent experiments. (D) Rb influx versus pH in SS RBCs. Rb influx was measured in fresh cells washed and incubated in HBS at various pH values. The acid-stimulated Rb influx is greater than 95% CI dependent and reflects KCC activity. When present, urea concentration was 600 mM (▴). Data are from a single experiment, representative of 3.