β3-adrenergic agonist counters oxidative stress and Na⁺-K⁺ pump inhibitory S-glutathionylation of placental cells: implications for preeclampsia

Oxidative stress from placental ischemia/reperfusion and hypoxia/reoxygenation (H/R) in preeclampsia is accompanied by Na⁺-K⁺ pump inhibition and S-glutathionylation of its β1 subunit (GSS-β1), a modification that inhibits the pump. β3-adrenergic receptor (β3-AR) agonists can reverse GSS-β1. We examined the effects of the agonist CL316,243 on GSS-β1 and sources of H/R-induced oxidative stress in immortalized first-trimester human trophoblast (HTR-8/SVneo) and freshly isolated placental explants from normal-term pregnancies. H/R increased GSS-β1 and, reflecting compromised α1/β1 subunit interaction, reduced α1/β1 pump subunit coimmunoprecipitation. H/R increased p47<i>^phox</i>/p22<i>^phox</i> NADPH oxidase subunit coimmunoprecipitation, reflecting membrane translocation of cytosolic p47<i>^phox</i> that is needed to activate NADPH oxidase. Fluorescence of O₂^•--sensitive dihydroethidium increased in parallel. H/R increased S-glutathionylation of endothelial nitric oxide synthase (GSS-eNOS) that uncouples nitric oxide synthesis toward the synthesis of O₂^•- and reduced trophoblast migration. Oxidative stress induced by tumor necrosis factor α increased soluble fms-like tyrosine kinase receptor 1 (sFlt-1) trophoblast release, a marker of preeclampsia, and reduced trophoblast integration into endothelial cellular networks. CL316,243 eliminated H/R-induced GSS-β1 and decreases of α1/β1 subunit coimmunoprecipitation, eliminated NADPH oxidase activation and increases in GSS-eNOS, restored trophoblast migration, eliminated increased sFlt-1 release, and restored trophoblast integration in endothelial cell networks. H/R-induced GSS-β1, α1/β1 subunit coimmunoprecipitation, and NADPH oxidase activation of placental explants reflected effects of H/R for trophoblasts and CL316,243 eliminated these changes. We conclude a β3-AR agonist counters key pathophysiological features of preeclampsia in vitro. β3 agonists already in human use for another purpose are potential candidates for repurposing to treat preeclampsia.<b>NEW & NOTEWORTHY</b> H/R-induced oxidative stress and deficient NO-dependent placentation are features of preeclampsia, yet nonspecific antioxidants and NO donors are ineffective. Here, activation of the microdomain-confined signaling pathway with an agonist for the eNOS-coupled β3-AR eliminates inhibitory glutathionylation of the Na⁺-K⁺ pump's β1 subunit, uncoupling of eNOS, and activation of NADPH oxidase that are sources of H/R-induced oxidative stress. The agonist also eliminates H/R-induced inhibition of trophoblast migration and their integration into an endothelial network.