Effect of DTT treatment on urea stimulation of KCC and volume reduction in reticulocytes. Fresh cells were washed and preincubated 1 hour without (control, filled symbols) or with 10 mM DTT (open symbols) as described in “Materials and methods,” under “Dithiothreitol treatment.” Points are mean ± SEM (n = 4); curves were drawn by eye. A parallel set of experiments with AA RBCs ± DTT are represented by a dashed line drawn by eye to fit the data points, which were omitted for clarity. (A) Rb influx measured at various urea concentrations in HBS pH 7.4. Parallel measurements in cells swollen to CHCM less than 270 g/L (27 g/dL) by nystatin treatment (± DTT pretreatment) provided a measure of maximal volume stimulated flux for normalizing data. Data are means ± SEM from 3 experiments independent from Figure 1. DTT pretreatment had no effect on urea stimulation of KCC in AA RBCs; data from cells incubated with and without DTT were combined to calculate the dashed line representing AA cells. (B) Effect of DTT and urea on volume reduction in SS reticulocytes. Unswollen cells were incubated as in Figure 4 at indicated urea concentrations, either without (filled symbols) or with (open symbols) DTT pretreatment. CHCM at 120 minutes was lower with DTT treatment at both 300 and 600 mM urea (P < .003 and P < .03, respectively, n = 3).