Uneven distribution of nucleoside transporters and intracellular enzymatic degradation prevent transport of intact [14C] adenosine across the sheep choroid plexus epithelium as a monolayer in primary culture

Abstract

Background: Efflux transport of adenosine across the choroid plexus (CP) epithelium might contribute to the homeostasis of this neuromodulator in the extracellular fluids of the brain. The aim of this study was to explore adenosine transport across sheep CP epithelial cell monolayers in primary culture.

Methods: To explore transport of adenosine across the CP epithelium, we have developed a method for primary culture of the sheep choroid plexus epithelial cells (CPEC) on plastic permeable supports and analysed [14C] adenosine transport across this cellular layer, [14C] adenosine metabolism inside the cells, and cellular uptake of [14C] adenosine from either of the chambers. The primary cell culture consisted of an enriched epithelial cell fraction from the sheep fourth ventricle CP and was grown on laminin-precoated filter inserts.

Results and conclusion: CPEC grew as monolayers forming typical polygonal islands, reaching optical confluence on the third day after the seeding. Transepithelial electrical resistance increased over the time after seeding up to 85 +/- 9 Omega cm2 at day 8, while permeability towards [14C] sucrose, a marker of paracellular diffusion, simultaneously decreased. These cells expressed some features typical of the CPEC in situ, including three nucleoside transporters at the transcript level that normally mediate adenosine transport across cellular membranes. The estimated permeability of these monolayers towards [14C] adenosine was low and the same order of magnitude as for the markers of paracellular diffusion.However, inhibition of the intracellular enzymes, adenosine kinase and adenosine deaminase, led to a significant increase in transcellular permeability, indicating that intracellular phosphorylation into nucleotides might be a reason for the low transcellular permeability. HPLC analysis with simultaneous detection of radioactivity revealed that [14C] radioactivity which appeared in the acceptor chamber after the incubation of CPEC monolayers with [14C] adenosine in the donor chamber was mostly present as [14C] hypoxanthine, a product of adenosine metabolic degradation. Therefore, it appears that CPEC in primary cultures act as an enzymatic barrier towards adenosine. Cellular uptake studies revealed that concentrative uptake of [14C] adenosine was confined only to the side of these cells facing the upper or apical chamber, indicating uneven distribution of nucleoside transporters.

Figure 1

Morphology and phenotype of primary culture of sheep CPEC. (A) Phase-contrast micrographs of 8d-old CPE cells cultured on laminin-coated filters shows a typical cobblestone arrangement of polygonal cells (scale bar 20 μm); (B) Scanning electron microscopy shows a confluent 8d-old monolayer of CPEC, with a prominent nuclei (scale bar 10 μm). (C) Changes with time of TEER in CPEC monolayers seeded on inserts pre-coated with various basal lamina components or on uncoated inserts. Note the marked increase in TEER between days 3 and 8 across the CPEC monolayers seeded on inserts pre-coated with laminin and across CPEC monolayers seeded on uncoated inserts. Values are expressed as mean ± SEM from 3–4 different filters and were corrected for the mean TEER from three laminin-coated filters without cells.

Figure 2

(A) The immunofluorescence assay using anti-cytokeratin antibodies showing a positive staining of the 8d-old monolayer of the CPEC (scale bar 10 μm). (B) Transmission electron micrographs of cultured CPE cells demonstrating the ultrastructural features of a polarized epithelial cell monolayer such as a tightly apposed lateral membrane with complex apical junctions organized as a tight junction (TJ) association and complex lateral interdigitations (LI) at the basolateral side (scale bar 0.01 μm, magnification 125.000 ×). (C) Scanning electron micrograph showing a number of processes on the CPEC side which faced apical (upper) chamber (scale bar 2 μm, magnification 6000 ×). (D) Eight-day-old CPE cells grown on laminin-coated filters were stained with primary antibodies against occludin and then with FITC conjugated secondary antibodies. A continuous circumferential distribution of fluorescence consistent with the establishment of TJs in CPEC monolayers is shown. Scale bar 20 μm.

Figure 3

A plot of the TEER across CPEC monolayers against the permeability to [¹⁴C] sucrose. All 21 measurements were made on confluent monolayers from day 5 to day 8 after the seeding. TEER across laminin-coated inserts, which were kept under the same conditions, was subtracted as the background. The points revealed a strong linear reverse proportion between these two parameters, with Pearson's quotient -0.847.

Figure 4

(A). Expression of transthyretin in CPEC cells at the transcript level. Total mRNA was isolated from sheep 4 V whole CP, EECF and CPEC in primary culture at 48 h, 72 h and 8d after seeding. The 436-bp-long and 332-bp-long fragments amplified using oligonucleotides specific for transthyretin and GAPDH, respectively, were visible in samples from both fresh tissue and cellular cultures at various periods after seeding. From these gels the expression of TTR was estimated relative to GAPDH and the values presented in the bar graphs. They show that TTR mRNA expression is similar to that of fresh tissue in 8d-old cultured cells. Sheep liver and heart homogenate were processed in parallel as positive and negative controls, respectively (not shown). Far left lane contains DNA molecular weight markers. (B) Expression of transthyretin in 8d-old monolayers of CPEC cells at the protein level. CPEC were treated with goat anti-transthyretin polyclonal IgG (1:400) and then with mouse anti-goat IgG. There was intensive fluorescence in the cytoplasm, while the nuclei were not stained. Scale bar 10 μm.

Figure 5

The expression of (A) CNT2, (B) ENT1, and (C) ENT2 at the transcript level in the 8d-old CPEC in primary culture and in the fresh EECF. All gels revealed bands corresponding to these nucleoside transporters in both fresh EECF and CPEC in primary culture. They were also present in samples from whole brain homogenate (positive control, not shown in the figure). The amount of mRNA for these proteins was expressed relative to the amount of mRNA for the housekeeping protein GAPDH and the mean values are presented as bars. The apparent amount of mRNA for GPADH did not differ between fresh EECF samples and samples from CPEC in primary culture. The relative expression of mRNA for ENT1 was the same in CPEC in primary culture as in EECF; however, relative expressions of mRNA for two other proteins were 40–50% lower in CPEC than in EECF.

Figure 6

Clearance of [¹⁴C] adenosine and [³H] mannitol across the 8d-old CPEC monolayers. (A) The donor was the upper chamber. (B) The donor was the lower chamber. Experiments were performed on CPEC monolayers from at least 2 animals. The concentration of [¹⁴C] adenosine was 125 nM, which represented about 5% of Km of [³H] adenosine uptake by the basolateral side of sheep CPEC in situ (I. Markovic, PhD Thesis, University of Belgrade, 1998). The clearance was linear; however, the values obtained were quite close to the values of [³H] mannitol clearance. Using these data points the permeability of CPEC monolayers for [¹⁴C] adenosine was calculated and these values are presented in Table 1.

Figure 7

HPLC-radiodetector analysis of standard, which represented the uptake buffer from (A) the donor chamber containing [¹⁴C] adenosine prepared as described in Methods section and (B) of the uptake buffer from acceptor chamber collected after 10 min of incubation with CPEC monolayers. Y-axis shows DPM, and retention time in minutes is on the X-axis. Under these conditions, peak elution of radioactivity in the standard occurred at 9.28 min. However, a negligible amount of radioactivity was eluted at this time in the sample from the acceptor chamber (B), where about 2/3 DPM appeared in the hypoxanthine peak and the rest in the adenine peak. These peaks were identified by retention times and spectral analysis.

Figure 8

Uptake of [¹⁴C] adenosine into primary cultured sheep CPEC. The panel shows the cellular uptake from the apical (upper) chamber as a donor (left side) and from the basolateral (lower) chamber as a donor (right). Values shown represent uptake of adenosine, corrected for tracer trapped in the extracellular space, after 5 or 10 min of incubation in the uptake buffer containing [¹⁴C] adenosine and [³H] mannitol in the donor chamber. Data are shown for uptake under control conditions in Na⁺-containing medium (black bars), using Na⁺-free uptake buffer (grey bars), or in uptake buffer containing both Na⁺ and 1 uM NBTI (open bars). All values are presented as mean ± SEM from three to five separate inserts obtained from at least two separate isolations. Statistical significance: n.s., P > 0.05 vs. control; **P < 0.01 vs. control by ANOVA.