Figure 7.
Human sperm display functional ENaCs sensitive to amiloride. A, human sperm proteins were analyzed by 8% SDS-PAGE and immunoblotted using antibodies against the β subunit of ENaC. The lane contained 5 × 106 sperm. B, amiloride (1 μm) decreased the concentration of [Na+]i assessed by CoroNa Red fluorescence and flow cytometry. C, the ENaC inhibitor amiloride (1 μm) rescued the inhibitory effect of S0859 (5 μm). D, fluorescence median of DISC3(5) shown in C was normalized with respect to CAP condition. Values represent the mean ± S.E.; ***, p < 0.001 (n = 5). E, human sperm were incubated in capacitating conditions (CAP) in the presence or absence of 1 μm amiloride, 5 μm S0859, or 5 μm inh-172. Then, the cells were loaded with DISC3(5) to evaluate Em. DISC3(5) fluorescence median was registered before (time = 0 min) and after (black arrow) addition of 40 mm NaCl. In each experiment, the fluorescence median was relativized to maximum fluorescence reached between 4 min addition of 1 μm valinomycin. Values represent the mean ± S.E. of at least 3 experiments. Two-way ANOVA with Dunnett's post test. ****, p < 0.0001 versus CAP at 4 min.