Regulation of the Endogenous Blood-Brain Barrier Transporter Organic Anion Transporting Polypeptide 1A4 (Oatp1a4) by Testosterone in an Immortalized Mouse Brain Endothelial Cell Line (bEnd.3)

Abstract

The biochemical and physical properties of the blood-brain barrier (BBB) are known to regulate drug delivery to the central nervous system (CNS), making it incredibly challenging to treat neurological diseases. A viable strategy may be to target organic anion transporting polypeptides (OATPs in humans; Oatps in rodents), transporters that facilitate blood-to-brain drug uptake. Over the past several years, our laboratory has studied the involvement of OATPs/Oatps in the BBB transport of drugs that are effective in treatment of neurological pathologies such as cerebral hypoxia/reoxygenation stress and ischemic stroke. Using male and female Sprague-Dawley rats, we have shown that Oatp1a4, the primary drug transporting Oatp isoform at the rodent BBB, is critical for brain delivery of 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase inhibitors (i.e., statins). More recently, we have shown that Oatp1a4 functional expression is higher at the BBB in female Sprague-Dawley rats as compared to their male counterparts. Interestingly, this work also showed that Oatp1a4 protein expression and transport activity in brain micro-vessels from castrated male rats was the same as in female control rats or in ovariectomized females. This observation pointed towards a role for male gonadal sex hormones in the regulation of Oatp1a4 at the BBB. Therefore, we sought to determine the effect of testosterone on Oatp1a4 protein expression using a mouse brain micro-vessel endothelial cell line (bEND.3). Specifically, we studied the effect of testosterone in normoxic cells and in cells subjected to oxygen/glucose deprivation (OGD), an in vitro condition relevant to ischemic stroke. In normoxic (i.e., control) bEND.3 cultures, testosterone increased Oatp1a4 protein expression in a dose-dependent manner. In contrast, testosterone reduced Oatp1a4 protein expression in bEND.3 cells subjected to 8 h OGD but had no effect in these cultured mouse brain endothelial cells after 8 h OGD/24 h reoxygenation (i.e., OGD/R). Interestingly, testosterone treatment increased expression of the androgen receptor under both OGD and OGD/R conditions. Overall, these data provide the first evidence for differential regulation of Oatp1a4 protein expression by testosterone under normoxic, OGD, and OGD/R conditions. Further studies are required to evaluate the implications of these findings on transport of Oatp1a4 substrates (i.e., statins) and to determine the molecular machinery involved in altered Oatp1a4 expression in endothelial cells following exposure to testosterone.