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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), 14 C-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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Disruption of blood-brain barrier integrity and dramatic failure of brain ion homeostasis including fluctuations of pH occurs during cortical spreading depression (CSD) events associated with several neurological disorders, including migraine with aura, traumatic brain injury and stroke. NHE1 is the primary regulator of pH in the central nervous system. The goal of the current study was to investigate the role of sodium-hydrogen exchanger type 1 (NHE1) in blood brain barrier (BBB) integrity during CSD events and the contributions of this antiporter on xenobiotic uptake. Using immortalized cell lines, pharmacologic inhibition and genetic knockdown of NHE1 mitigated the paracellular uptake of radiolabeled sucrose implicating functional NHE1 in BBB maintenance. In contrast, loss of functional NHE1 in endothelial cells facilitated uptake of the anti-migraine therapeutic, sumatriptan. In female rats, cortical KCl but not aCSF selectively reduced total expression of NHE1 in cortex and PAG but increased expression in trigeminal ganglia; no changes were seen in trigeminal nucleus caudalis. Thus, in vitro observations may have a significance in vivo to increase brain sumatriptan levels. Pharmacological inhibition of NHE1 prior to cortical manipulations enhanced the efficacy of sumatriptan at early time-points but induced facial sensitivity alone. Overall, our results suggest that dysregulation of NHE1 contributes to breaches in BBB integrity, drug penetrance, and the behavioral sensitivity to the antimigraine agent, sumatriptan., Competing Interests: The authors have declared that no competing interests exist.

In the brain, insulin acts as a growth factor, regulates energy homeostasis, and is involved in learning and memory acquisition. Many central nervous system (CNS) diseases are characterized by deficits in insulin signaling. Pre-clinical studies have shown that intranasal insulin is neuroprotective in models of Alzheimer's disease, Parkinson's disease, and traumatic brain injury. Clinical trials have also shown that intranasal insulin elicits beneficial cognitive effects in patients with Alzheimer's disease. It is known that insulin can be detected in the CNS within minutes following intranasal administration. Despite these advances, the anatomical pathways that insulin utilizes to reach the CNS and the cellular CNS targets after intranasal administration are not fully understood. Here, we intranasally administered fluorescently labeled insulin and imaged its localization within the brain and trigeminal nerves. Our data indicates that intranasal insulin can reach cellular CNS targets along extracellular components of the trigeminal nerve. Upon CNS entry, we found insulin significantly increased levels of an activated form of the insulin receptor. These findings suggest that the intranasal route of administration is able to effectively deliver insulin to CNS targets in a biologically active form.

P-glycoprotein (PgP) is the major drug efflux pump in human cerebral microvessels. PgP prevents pathogens, toxins and therapeutic drugs from entering the CNS. Understanding the molecular regulation of PgP activity will suggest novel mechanisms to improve CNS drug delivery. Previously, we found that during peripheral inflammatory pain (PIP) (3 h after λ carrageenan injection in the rat paw), PgP traffics to the cortical microvessel endothelial cell plasma membrane concomitant with increased PgP activity. In the current study, we measured the changes in composition of PgP-containing protein complexes after PIP in rat microvessel isolates. We found that a portion of the PgP is contained in a multi-protein complex that also contains the caveolar proteins CAV1, SDPR, PTRF and PRKCDBP. With PIP, total CAV1 bound to PgP was unchanged; however, phosphorylated CAV1 (Y14P-CAV1) in the complex increased. There were few PgP/CAV1 complexes relative to total PgP and CAV1 in the microvessels suggesting CAV1 bound to PgP is unlikely to affect total PgP activity. However, both PgP and CAV1 trafficked away from the nucleus in response to PIP. These data suggest that P-CAV1 bound to PgP potentially regulates PgP trafficking and contributes to the acute PgP activity increase after a PIP stimulus.