Interactive effects of in vitro binge-like alcohol and ATP on umbilical endothelial nitric oxide synthase post-translational modifications and redox modulation

• Chronic in vitro binge alcohol exposure dysregulates the exquisite coordination and regulation of umbilical endothelial vascular adaptations.
• Binge-like alcohol has detrimental effects on all four major eNOS multi-site phosphorylation sites.
• There is an interactive effect of alcohol and the endogenous eNOS activating agonist ATP on eNOS multi-site phosphorylation state.
• Alcohol modulates eNOS-related redox switch post-translational modifications.
• Major role for the reproductive vascular system in pathogenesis of Fetal Alcohol Spectrum Disorders.

Alcohol dysregulates the regulation of reproductive vascular adaptations. We herein investigated chronic in vitro binge-like alcohol effects on umbilical endothelial nitric oxide synthase (eNOS) multi-site phosphorylation and related redox switches under basal (unstimulated) and stimulated (with ATP) states. Alcohol decreased endothelial excitatory Pser1177eNOS (P < 0.001), whereas excitatory Pser635eNOS exhibited a main effect of alcohol (↓P = 0.016) and ATP (↑P < 0.001). Alcohol decreased Pthr495eNOS (P = 0.004) levels, whereas inhibitory Pser116eNOS exhibited an alcohol main effect in both basal and stimulated states (↑P = 0.005). Total eNOS was reduced by alcohol (P = 0.038). In presence of ATP, alcohol inhibited ERK activity (P = 0.002), whereas AKT exhibited no alcohol effect. Alcohol main effect on S-nitroso-glutathione reductase (↓P = 0.031) and glutathione-S-transferase (↓P = 0.027) were noted. Increased protein glutathiolation was noted, whereas no alcohol effect on GSH, GSSG, NOX2 or SOD expression was noted. Thus, alcohol effects on multi-site post-translational modifications and redox switches related to vasodilatory eNOS underscore the necessity for investigating alcohol-induced gestational vascular dysfunction.