Fig. 4.

Effect of dimethyl adipimidate on sickling and K⁺ transport in Mg²⁺ clamped human sickle red cells. HbS cells samples were exposed to dimethyl adipimidate (6 mM) for 30 min at 37°C, pH 8, then washed three times to remove unreacted DMA; control samples were similarly treated but without DMA. Both were then placed in tonometers (40% haematocrit) and equilibrated with O₂ at 150 mmHg or 100% N₂. Cells were subsequently Mg²⁺ clamped, as described in the legend to Figure 1, except that only a single [Mg²⁺]_o, 0.19 mM (plus 50μM EGTA), was used. Aliquots were removed, fixed in saline with 1% glutaraldehyde (pre-equilibrated with air or N₂, as appropriate) for assessment of cell shape using light microscopy. Finally, K⁺ influxes were measured: Cl⁻-dependent K⁺ influx was taken as a measure of the activity of K⁺-Cl⁻ cotransporter; the deoxygenation-induced Cl⁻ independent K⁺ influx as a measure of P_sickle activity. Ouabain (100 μM) and bumetanide (10 μM) were present. Data for K⁺ transport pathways or cell sickling are presented for cells in N₂, normalised to 100% in the absence of DMA. Histograms represent means ± S.E.M. for 3 determinations.