Live tissue imaging of organic cation and anion transport at the blood‐CSF barrier reveals specific transporter function and distinct transcellular pathways

Abstract

Formed by the choroid plexus (CP) epithelial cells, the blood‐cerebrospinal fluid (CSF) barrier plays an active role in removing neurotoxins, drugs, and metabolites from the brain. Several organic cation and anion transporters, including the plasma membrane monoamine transporter (PMAT), organic anion transporter 1 and 3 (OAT1/3), and multidrug resistance‐associated protein 1 and 4 (MRP1/4) have been reported to be expressed in CP, but their relative expression and specific function at the blood‐CSF barriers have not been well characterized at the tissue level. The goal of this study is to quantify the expression of organic cation and anion transporters in CP tissues and determine their specific roles in organic cation or anion transport by live tissue imaging using fluorescent substrates. In both human and mouse CPs, real‐time PCR analysis showed highest mRNA expression of PMAT followed by MRP1, OAT3 and MRP4. CP also expresses several drug metabolizing enzymes including the monoamine oxidases (MAOs), catechol‐O‐methyltransferase (COMT), UDP‐glucuronosyltransferases (UGTs) and sulfotransferases (SULT). In CP isolated from wildtype (wt) mouse, the fluorescent organic cation probe IDT307 is transported into CP cells at the apical (CSF‐facing) membrane and is highly accumulated in CP cells with a large storage in mitochondria. There was little efflux at the basolateral membrane. In contrast, IDT307 uptake and intracellular accumulation is attenuated by ~3–4 folds in CPs isolated from Pmat knockout mice. Interestingly, in wt CPs, the organic anion probe fluorescein‐methotrexate (FL‐MTX) is rapidly taken up and efficiently excreted into the capillary side with little intracellular accumulation. MK571, a potent inhibitor of MRP1/4, abolished basolateral efflux of FL‐MTX, resulting in high accumulation within CP cells. Together, our study in live CP tissue demonstrates an important role of PMAT in apical uptake of organic cations and a crucial function of MRP1/4 in basolateral efflux of organic anions. Based on these studies, we propose distinct molecular and cellular pathways for organic cation and anion transport at the blood‐CSF barrier.Support or Funding InformationThis study is supported by National Institutes of Health Grant GM066233.