Figure 5.

Treatment with CysNO leads to S-nitrosation of spectrin and protects RBCs from t-BuOOH-induced impairment of RBC deformability. (A) S-nitrosation of spectrin following treatment of RBC suspension with 10⁻⁹ to 10⁻² M CysNO for 30 min as assessed by biotin switch assay (upper lane). Total spectrin, on the same membrane determined by Western blot. Each lane is labeled with the treatment concentration of CysNO. Lane MWM is the corresponding molecular weight marker (for whole images of whole membrane please refer to Figure S4). (B) Increasing concentrations of CysNO did not affect RBC deformability, except at the highest concentration (50,000 μM CysNO). Two-way RM ANOVA p = 0.0059 and Dunnett's test vs. untreated control, ^*p ≤ 0.05 (for 50,000 μM statistically significant over a range of shear stresses from 0.53 to 53.33 Pa). (C) Pre-incubation of RBCs with different concentrations of nitrosating agent CysNO significantly rescued RBC deformability after treatment with 3 mM t-BuOOH (n = 5). Two-way RM ANOVA p < 0.0003 and Tukey's test; ^*p ≤ 0.05 vs. 3 mM t-BuOOH (statistically significant for 1 μM CysNO pre-incubation in the shear stresses from 5.33 to 53.33 Pa and for 1000 μM CysNO pre-incubation in the shear stress range from 3 to 53.33 Pa). (D) Post-incubation of RBCs with different concentrations of nitrosating agent CysNO significantly rescued RBC deformability after treatment with 3 mM t-BuOOH (n = 5). Two-way RM ANOVA p < 0.0003 and Tukey's test; ^*p ≤ 0.05 vs. 3 mM t-BuOOH (statistically significant for 1 μM CysNO and 1000 μM CysNO in the range of shear stresses from 5.33 to 53.33 Pa). Deformability index of RBC pellets measured in a range of shear stresses of 0.3–53.33 Pa.