Fig. 4. NO and protein S-nitrosylation in sperm. (A) 100 µM GSNO, an S-nitrosylating agent, causes a rise in [Ca²⁺]_i similar to that seen with NONOate but the onset of the effect is more rapid. Responses of six cells are shown. (B) 100 µM GSH rapidly reverses the action of 100 µM GSNO on sperm [Ca²⁺]_i. Responses of five cells are shown. (C) GSNO causes rapid S-nitrosylation of sperm proteins: lane 1 shows background levels in cells processed immediately for assay (indicated by ^*); lane 2 shows that, after 60 minutes of incubation of the cells in sEBSS, this level does not change; lanes 3, 4, 5, 6 and 7 show increased S-nitrosylation in cells processed for assay immediately upon exposure to 50 µM GSNO (^*), and those incubated with GSNO for 5, 10, 30 and 60 minutes, respectively. S-nitrosylation reaches near steady-state levels in the sample processed immediately (5 minutes for preliminary centrifugation; see Materials and methods). (D) S-nitrosylation of sperm proteins is rapidly reversible. Left panel shows S-nitrosylated proteins in untreated cells incubated for 10 minutes (lane 1), cells exposed to GSNO and cys-SNO (lanes 2 and 4), and cells exposed to GSH and exhausted cys-NO (lanes 3 and 5; controls). Right panel shows same treatments but cells were washed in PBS immediately before processing for the assay. S-nitrosylation caused by GSNO and CSNO is rapidly reversed upon removal of the agent.