ObjectivesBinding of nitric oxide (NO) to a cysteine thiol is termed S‐nitrosylation, which is emerging as an important means of NO‐mediated signal transduction independent of stimulation of soluble guanylate cyclase. However, the impact of S‐nitrosylation on erectile function remains unclear. S‐nitrosylation is a redox sensitive post‐translational modification, which is thought to be promoted by reactive oxygen species (ROS). Protein S‐nitrosylation levels are regulated in part by S‐nitrosoglutatione reductase (GSNOR), which acts to de‐nitrosylate protein thiols. The objectives of this study were to determine the role of GSNOR on maintenance of redox balance and erectile function in response to a Western diet (WD) high in fat and sugar.Materials and MethodsYoung male wild type (WT; n = 110) and GSNOR−/− (n = 108) mice were fed either a control diet or a WD ad libitum for 3, 6, 9, or 12 weeks. Following the dietary intervention, erectile function was assessed by measuring intracavernosal pressure (ICP) and mean arterial pressure (MAP) during cavernous nerve stimulation. In separate mice, in vivo ROS production was measured in the penis utilizing a novel microdialysis approach. Microdialysis probes were inserted into the penis of anesthetized mice and perfused with saline containing 100 uM Amplex Ultrared, 1 U/ml horseradish peroxidase, and 10 U/ml superoxide dismutase. ROS convert the reagents to a fluorescent byproduct resorufin, which was measured in the outflowing dialysate. Penile tissue was harvested from mice and processed for enrichment of nitrosylated thiols. Nitrosylated thiols were labeled with iodoTMT, and nitrosylated proteins were detected by mass spectrometry.ResultsNeither ROS generation (WT: 0.66 ± 0.09 vs. GSNOR−/− 0.96 ± 0.12 μM H2O2; p = 0.41) nor erectile function (WT:8.44 ± 1.4 vs. GSNOR−/− 9.6 ± 1.6 ICP area/MAP; p = 0.97) were different between genotypes fed the control diet. However, ROS were elevated following just 3 weeks of WD consumption in GSNOR−/− mice (1.44 ± 0.19; p = 0.01), whereas ROS increased following 6 weeks of WD in WT mice (1.29 ± 0.19; p = 0.02). Subsequent to increases in ROS, ED was evident after 6 weeks of WD in GSNOR−/− mice (5.0 ± 1.1, p = 0.04) and after 12 weeks of WD in WT mice (3.1 ± 0.6, p = 0.02). 249 unique peptides corresponding to 136 different proteins with nitrosylated thiol groups were identified by mass spectrometry.ConclusionsMice lacking GSNOR demonstrated an impaired ability to maintain redox balance in the face of the pro‐oxidant stimulus of the WD, prompting accelerated development of erectile dysfunction. These data implicate a protective role for GSNOR in the penis and suggest that excessive S‐nitrosylation may negatively regulate erectile function.Support or Funding InformationSupported by a mini grant from the National Kidney Foundation of Maryland and National Institutes of Health grant 1F32DK100082.