Functional characterisation of the active ascorbic acid transport into cerebrospinal fluid using primary cultured choroid plexus cells

Abstract

Crossing the blood–CSF barrier is an important pathway for certain nutrients to enter the CNS. Cultured choroid plexus epithelial cells are a potent model system to study active transport properties of this tissue in vitro. In the present study this in vitro model was used to analyse ascorbic acid transport across the blood–CSF barrier that is supposedly mediated by the Na⁺-dependent transporter SVCT2. The expression of SVCT2 in the cultured cells was proven by RT-PCR. Active transport across the cell monolayer resulted in ascorbic acid enrichment at the CSF mimicking side. Ascorbic acid transport and uptake were decreased to 13 and 27%, respectively, in the presence of 200 μM phloretin. Inhibition of both transepithelial substrate transport (to 7.5%) and cytoplasmatic uptake (to 20%) was observed in Na⁺-free medium indicating that a basolaterally located and Na⁺-dependent transporter mediates ascorbic acid uptake. Substituting Cl⁻ by either iodide or d-gluconate increased ascorbic acid uptake by factors of 3.7 or 2.5, respectively. Similar observations were made when Na⁺-dependent myo-inositol transport was analysed. Additionally, in presence of 100 μM bumetanide, an inhibitor of Na⁺-Cl⁻-cotransport, indirectly increased ascorbic acid and myo-inositol transport rates were observed showing that ascorbic acid-Na⁺-cotransport might balance low intracellular Na⁺ concentration.

Introduction

Two barrier systems, the blood–brain barrier and the blood–CSF barrier, serve to maintain brain homeostasis by restricting uncontrolled diffusion of water-soluble molecules into CNS. For proper neural function macronutrients and micronutrient are required that enter the brain either by facilitated diffusion or active transport, mediated by specific transport systems. The blood–CSF barrier forming epithelial cells of the choroid plexus (CP) are supposed to play an important role only in micronutrient homeostasis [20], e.g., in vitamin C transport into CNS [23]. Great amounts of ascorbic acid (AA), the reduced form of vitamin C, are enriched in the brain. AA concentration in the cerebrospinal fluid (CSF) is approximately 200 μM and thus about four times higher than typical serum concentrations of 50 μM [17], [24]. In neural tissue AA concentration even reaches millimolar ranges, depending on cell type and regional distribution as reviewed by Rice [18]. The redox characteristics of AA enable this molecule to act as an important enzyme cofactor in metabolic reactions and as a trap for free radicals protecting tissues from oxidative damage. Within the CNS, AA is involved in neurotransmitter and hormone synthesis as a cofactor for dopamine-β-hydroxylase and peptidylglycine α-amidating monooxygenase [5], [8], in myelin formation by stimulating Schwann-cell differentiation [6] and as an antioxidant it has neuroprotective functions.

In CP epithelium the localisation of the recently cloned AA specific transporter SVCT2 was proven by in situ hybridisation [25]. Daruwala et al. [3] demonstrated for transfected Xenopus oocytes, that via the SVCT2 system, AA transport is coupled to a cotransport of two Na⁺. A Na⁺ gradient established by the Na⁺–K⁺-ATPase is the driving force of this process.

A second pathway for vitamin C to enter CNS is to pass the endothelial cells of the brain microvessels in its oxidised form dehydroascorbic acid (DHA) via the glucose transporter GLUT1 [1]. The existence of the first pathway is underlined by the facts that vitamin C is circulating in the blood in its reduced form most of its lifetime [4] and that it has to be transported against a steep concentration gradient into the CNS. Although some groups consider the second pathway to be prominent, vitamin C transport by GLUT 1 seems unlikely in the presence of physiological glucose plasma concentrations. Further investigations are necessary to quantify the uptake routes of vitamin C into CNS for both routes.

It has also been shown that myo-inositol (MI) is actively transported by the CP epithelium and is enriched in the CSF [21], [22]. The mechanism and regulation of the MI-Na⁺-cotransporter SMIT that specifically promotes transepithelial MI transport has been demonstrated before [13], [15].

In earlier studies the barrier and transport properties of primary cultured epithelial cells of porcine CP have been investigated. The cells form confluent monolayers within a few days after seeding them on permeable filter membranes. They establish high transepithelial electrical resistances (TER) of about 1000–1700 Ω·cm² after removal of serum from the medium and actively transport fluid from the basolateral to the apical chamber [9]. AA and MI are transported from the basolateral to the apical side in a concentration-dependent process [10]. The K_M values for the transport of AA (67 μM) and MI (117 μM) are in good agreement to data derived from uptake measurements with CP tissue [21], [23] and SVCT2-cDNA transfected HRPE-cells [16]. We concluded that this cell culture model closely mimics the in vivo situation and can be applied to study transport characteristics of blood–CSF barrier in vitro, alternatively to tissue or in vivo experiments.

Here we report for the first time that cultured CP epithelial cells as a model of the blood–CSF barrier are well suited to investigate AA transport mechanism. From inhibition studies we conclude that AA is taken up by a Na⁺-dependent transport system as it has been shown for SVCT2 mediated transport. Additionally we demonstrate the transporter’s localisation within the basolateral membrane. In several experiments we have studied the inhibition and activation of the transporter and its interactions with other transport systems depending on ion concentration gradients.