Sex hormones regulate NHE1 functional expression and brain endothelial proteome to control paracellular integrity of the blood endothelial barrier

Background: Sex hormones have been implicated in pH regulation of numerous physiological systems. One consistent factor of these studies is the sodium-hydrogen exchanger 1 (NHE1). NHE1 has been associated with pH homeostasis at epithelial barriers. Hormone fluctuations have been implicated in protection and risk for breaches in blood brain barrier (BBB)/blood endothelial barrier (BEB) integrity. Few studies, however, have investigated BBB/BEB integrity in neurological disorders in the context of sex-hormone regulation of pH homeostasis. Methods//Results: Physiologically relevant concentrations of 17-β-estradiol (E2, 294 pM), progesterone (P, 100 nM), and testosterone (T,3.12 nM) were independently applied to cultured immortalized bEnd.3 brain endothelial cells to study the BEB. Individual gonadal hormones showed preferential effects on extracellular pH (E2), ¹⁴C-sucrose uptake (T), stimulated paracellular breaches (P) with dependence on functional NHE1 expression without impacting transendothelial resistance (TEER) or total protein expression. While total NHE1 expression was not changed as determined via whole cell lysate and subcellular fractionation experiment, biotinylation of NHE1 for surface membrane expression showed E2 reduced functional expression. Quantitative proteomic analysis revealed divergent effects of 17-β-estradiol and testosterone on changes in protein abundance in bEnd.3 endothelial cells as compared to untreated controls. Conclusions: These data suggest that circulating levels of sex hormones may independently control BEB integrity by 1) regulating pH homeostasis through NHE1 functional expression and 2) modifying the endothelial proteome.