Fig. 5. Thiol reducing agents reverse NO effects. (A) DTT rapidly reverses nitrosylation of sperm proteins. Lane 1 shows endogenous S-nitrosylation in cells incubated in sEBSS for 60 minutes. Lanes 2 and 3 show cells incubated in the presence of 1 mM GSH (control) and 100 µM GSNO. Lane 4 shows cells incubated as for lane 3 but 1 mM DTT was added to the incubation 5 minutes before processing for the assay. (B) DTT reverses the action of 100 µM spermine NONOate. Upon application of 1 mM DTT, the increase in fluorescence induced by spermine NONOate is rapidly reduced or completely reversed. Responses of five separate cells are shown. (C) The DTT-induced decrease in fluorescence is correlated with the preceding NONOate-stimulated increase in fluorescence. Scattergram shows data from a single experiment, representative of five repeats. R²=0.33. (D) The action of DTT is not due to e^--dependent mitochondrial Ca²⁺ accumulation. After application of 100 µM spermine NONOate to mobilise Ca²⁺, the cells were exposed to 10 µM CCCP to collapse the mitochondrial inner membrane potential. The effect of subsequent application of 1 mM DTT resembled that seen in cells with functioning mitochondria. Responses of five cells are shown.