Transport of sulphate, thiosulphate and iodide by choroid plexus in vitro

—Isolated choroid plexuses of rabbits and cats were incubated in artificial cerebrospinal fluid medium containing [³⁵S]sulphate, [³⁵S]thiosulphate or [¹²⁵I]iodide and combinations thereof. After 1 hr incubation the mean ratio of tissue concentration to medium concentration was 2·46 for [³⁵S]sulphate, 2·39 for [³⁵S]thiosulphate, and 270 for [¹²⁵I]iodide. Uptake of all three anions was greatly reduced at 0° and by addition of dinitrophenol to the medium. Other inhibitors selectively reduced the uptake of particular anions; non-radioactive sulphate and thiosulphate reduced both [³⁵S]sulphate and [³⁵S]-thiosulphate uptake with much less effect on [¹²⁵I]iodide uptake, while non-radioactive iodide and thiocyanate greatly reduced [¹²⁵]iodide uptake with little or no effect on [³⁵S]sulphate or [³⁵S]thiosulphate uptake. It was concluded: (a) that sulphate and thiosulphate, like iodide, were accumulated by choroid plexus in vitro by active transport; (b) that sulphate and thiosulphate share and compete for a transport mechanism which is separate from the iodide transport mechanism; and (c) that the transport of sulphate out of cerebrospinal fluid demonstrated in vivo could occur at least in part in the choroid plexus.     

Thyroxine transport in choroid plexus

The role of the choroid plexus in thyroid hormone transport between body and brain, suggested by strong synthesis and secretion of transthyretin in this tissue, was investigated in in vitro and in vivo systems. Rat choroid plexus pieces incubated in vitro were found to accumulate thyroid hormones from surrounding medium in a non-saturable process. At equilibrium, the ratio of thyroid hormone concentration in choroid plexus pieces to that in medium decreased upon increasing the concentration of transthyretin in the medium. Fluorescence quenching of fluorophores located at different depths in liposome membranes showed maximal hormone accumulation in the middle of the phospholipid bilayer. Partition coefficients of thyroxine and triiodothyronine between lipid and aqueous phase were about 20,000. After intravenous injection of 125I-labeled thyroid hormones, choroid plexus and parts of the brain steadily accumulated 125I-thyroxine, but not [125I]triiodothyronine, for many hours. The accumulation of 125I-thyroxine in choroid plexus preceded that in brain. The amount of 125I-thyroxine in non-brain tissues and the [125I]triiodothyronine content of all tissues decreased steadily beginning immediately after injection. A model is proposed for thyroxine transport from the bloodstream into cerebrospinal fluid based on partitioning of thyroxine between choroid plexus and surrounding fluids and binding of thyroxine to transthyretin newly synthesized and secreted by choroid plexus.     

Deoxycytidine transport and metabolism in choroid plexus

In vitro, the transport into and release of [3H]deoxycytidine from the isolated choroid plexus, the anatomical locus of the blood-cerebrospinal fluid barrier, were studied separately. By use of the ability of nitrobenzylthioinosine (NBTI) to inhibit deoxycytidine efflux from choroid plexus, the transport of 1 microM [3H]deoxycytidine into choroid plexus at 37 degrees C was measured. Deoxycytidine was transported into choroid plexus against a concentration gradient by a saturable process that depended on intracellular energy production, but not intracellular binding or metabolism. The Michaelis-Menten constant (KT) for the active transport of deoxycytidine into choroid plexus was 15 microM. The active transport system for deoxycytidine was inhibited by naturally occurring nucleosides and deoxynucleosides, but not by 1 mM probenecid and 2-deoxyribose or 100 microM cytosine and cytosine arabinoside. With less than 1 microM [3H]deoxycytidine in the medium, the choroid plexus accumulated [3H]deoxycytidine against a concentration gradient. However, approximately 50% of the [3H]deoxycytidine was phosphorylated to [3H]deoxycytidine nucleotides at a low extracellular [3H]deoxycytidine concentration (6 nM) in 15-min incubations. This accumulation process depended, in part, on saturable intracellular phosphorylation. These studies provide further evidence that the choroid plexus contains an active nucleoside transport system of low specificity for deoxynucleosides and ribonucleosides, and a separate, saturable efflux system for deoxynucleosides which is very sensitive to inhibition by NBTI.     

Folate transport in the choroid plexus.

The uptake of 5-methyltetrahydrofolic acid (5-MTHF) by the isolated choroid plexus of hog was studied and shown to be both temperature and time dependent. Uptake of 5-MTHF by the isolated choroid plexus was a saturable process and exhibited a K_t of 0.9 × 10⁻⁶M and Vmax of 1.39 nmole/gm dry wt/min. The system did not require the presence of sodium ion nor was it ouabain sensitive. The presence of metabolic inhibitors, e.g., 2,4-dinitrophenol, did not suppress the uptake rate. Deprivation of oxygen also did not affect the rate of 5-MTHF transport. Addition of folic acid to the incubating medium led to countertransport of intracellular 5-MTHF. Efflux studies also indicated that the majority of the intracellular 5-MTHF was rapidly exchangeable and therefore probably present in the cell water in a free state. Chromatographic analyses confirmed that 5-MTHF was not metabolically altered during the transport process. It is suggested that 5-methyltetrahydrofolic acid is transported in the isolated choroid plexus via a carrier-mediated process.     

Nucleoside transport in choroid plexus: Mechanism and specificity

In vitro the transport into and release of [³H]thymidine, [³H]deoxyuridine, and [³H]nitrobenzylthioinosine (NBTI) from the isolated choroid plexus, the anatomical locus of the blood-cerebrospinal fluid barrier, were studied separately. Using the ability of NBTI to inhibit nucleoside efflux from the choroid plexus, the transport of [³H]thymidine and [³H]deoxyuridine into the choroid plexus at 37 °C was measured. Like thymidine, deoxyuridine was transported into the choroid plexus against a concentration gradient by a saturable process that depended on intracellular energy production but not intracellular binding or metabolism. The Michaelis-Menten constants (K_T) for the active transport of thymidine and deoxyuridine into the choroid plexus were 13.6 and 7.2 μM, respectively. Deoxyuridine and adenosine were competitive inhibitors of thymidine transport into the choroid plexus with inhibitor constants (K_I) of 6.8 and 14.5 μM, respectively. [³H]NBTI was also transported into the choroid plexus at 37 °C; unlike [³H]thymidine and [³H]deoxyuridine, the release of [³H]NBTI was not inhibited by NBTI itself. These studies provide evidence that the choroid plexus contains an active nucleoside transport system of low specificity for nucleosides, and a separate, saturable efflux system for nucleosides that is very sensitive to inhibition by NBTI. In vivo these systems transport nucleosides from blood into cerebrospinal fluid.     

Amino acid transport by choroid plexus in vitro

Choroid plexus from mongrel cats was incubated from 1 to 120 min in artificial cerebrospinal fluid containing α-amino[1-¹⁴C]isobutyric acid. The uptake of α-amino [1-¹⁴C]isobutyric acid occurred against a concentration gradient, was saturable, dependent on metabolic energy, and inhibited by natural amino acids. These results indicate that a transport mechanism is present in choroid plexus which could serve to regulate amino acid concentration in the cerebrospinal fluid of animals.     

Transepithelial organic anion transport by shark choroid plexus

Spiny dogfish shark (Squalus acanthias) lateral and IV choroid plexuses (CPs) are ultrastructurally similar to the corresponding tissues of rat. However, shark IV CP is proportionally larger and easily accessible. Moreover, this epithelial sheet can be halved and studied in Ussing flux chambers. We have used confocal fluorescence microscopy and radiotracer techniques to characterize transepithelial transport of the organic anions (OAs) fluorescein (FL) and 2,4-dichlorophenoxyacetic acid (2,4-D), respectively, by shark CP. Lateral and IV CP accumulated 1 microM FL, with highest levels in the underlying extracellular spaces, intermediate levels in epithelial cells, and lowest levels in the medium. 2,4-D and probenecid inhibited FL accumulation in cells and extracellular spaces, suggesting that these substrates compete for common carriers. Unidirectional absorptive [cerebrospinal fluid (CSF)-to-blood] and secretory (blood-to-CSF) fluxes of 10 microM [(14)C]2,4-D were measured under short-circuited conditions in IV CP mounted in Ussing chambers. 2,4-D underwent net absorption, with an average flux ratio of 7. Probenecid, 2,4,5-trichlorophenoxyacetic acid, and 5-hydroxyindolacetic acid reduced net absorption, reversibly inhibiting unidirectional absorption, with no effect on secretion. Ouabain irreversibly reduced net 2,4-D absorption and cellular and extracellular accumulation of FL, suggesting energetic coupling of OA absorption to Na(+) transport. Collectively, these data indicate that shark CP actively removes OAs from CSF by a process that is specific and active.     

Uptake and transport of immunoglobulin by the chick choroid plexus

Summary The choroid plexus from the lateral ventricles of 18-day chick embryos was cultivated as an organ in medium 199 until the degeneration of the stroma. Selected plexuses forming an empty epithelial sac were then incubated with enzyme-treated human immunoglobulin (5S-antibody) and with native human immunoglobulin (7S-antibody). Uptake of the 7S-antibody was observed after 30 min, whereas the 5S-antibody was taken up by the choroid plexus within 1 min, as demonstrated by means of the peroxidase-anti-peroxidase (PAP) technique (Sternberger 1974). The antibodies were located in conspicuous, large vacuoles of the choroid epithelium. Further experiments were performed using only 5S-antibody. In addition to the demonstration of the protein structure of this immunoglobulin, it was also shown that its binding capacity for tetanus toxoid as an antigen remains intact in the intracellular location. It was not possible to observe lysosomal degradation. Moreover, 5S-antibody was detectable in cultures first incubated with 5S-antibody for 30 min and subsequently in antibody-free medium for a further period of 7 to 11 days.The biological significance of the uptake of material from the cerebrospinal fluid and the possibility of the existence of a receptor for 5S-antibody are discussed.     

Alteration of sodium transport by the choroid plexus with amiloride

Cerebrospinal fluid (CSF) production results from active transport of Na+ from blood to CSF, which is followed by H2O and anions. Amiloride reduces Na+ movement in epithelial tissues. To ascertain if amiloride alters transport of Na+ in the choroid plexus, the drug was administered either i.p. to male Sprague-Dawley rats that were bilaterally nephrectomized to determine in vivo effects, or added to artificial CSF to incubate the choroid plexus in vitro. Choroid cell [Na+] was reduced after amiloride treatment both in vivo and in vitro. In addition, the rate of 22Na uptake into the CSF and choroid plexus (CP) was decreased after amiloride. Alterations in choroid cell [Na+] and 22Na penetration into CSF and CP occurred at relatively high doses of drug (1 mumol/ml, in vitro and 100 micrograms/g in vivo), but lower doses were less effective (0.1 mumol/ml in vitro and 10 micrograms/g in vivo). It is concluded that the effects of amiloride on Na+ distribution and transport in the CP are due to inhibition of basolateral Na+-H+ exchange.     

Protein synthesis and transport by the rat choroid plexus and ependyma

Summary Light (LM-ARG) and electron microscope (EM-ARG) autoradiographs were prepared from immature rat choroid plexus and ependyma at 5, 10, 30, and 60 min and 16 h following intraperitoneal administration of [³H]- labeled amino acid mixtures. Intracellular protein synthesis and transport were ascertained in lateral and fourth ventricle choroid plexus epithelium by quantitative EM-ARG at the several post-injection intervals. ARG were also prepared from choroid plexuses cultured for one day, pulse labeled for one hour and reincubated for various periods in nonradioactive media. Significant labeling of both attached and free apical protrusions (blebs) was observed in both choroid plexus and ependyma in vivo and in choroid plexus in vitro. This phenomenon was interpreted as a physiologically significant mechanism for protein transport (apocrine secretion) by epithelia into the cerebrospinal fluid (CSF).This study was supported in part by N.I.H. Research Grant NS 12906     

Fluorescein-methotrexate transport in dogfish shark (Squalus acanthias) choroid plexus

The vertebrate choroid plexus removes potentially toxic metabolites and xenobiotics from cerebrospinal fluid (CSF) to blood for subsequent excretion in urine and bile. We used confocal microscopy and quantitative image analysis to characterize the mechanisms driving transport of the large organic anion, fluorescein-methotrexate (FL-MTX), from bath (CSF-side) to blood vessels in intact lateral choroid plexus from dogfish shark, Squalus acanthias, an evolutionarily ancient vertebrate. With 2 microM FL-MTX in the bath, steady-state fluorescence in the subepithelium/vascular space exceeded bath levels by 5- to 10-fold, and fluorescence in the epithelial cells was slightly below bath levels. FL-MTX accumulation in both tissue compartments was reduced by NaCN, Na removal, and ouabain, but not by a 10-fold increase in medium K. Certain organic anions, e.g., probenecid, MTX, and taurocholate, reduced FL-MTX accumulation in both tissue compartments; p-aminohippurate and estrone sulfate reduced subepithelial/vascular accumulation, but not cellular accumulation. At low concentrations, digoxin, leukotriene C4, and MK-571 reduced fluorescence in the subepithelium/vascular space while increasing cellular fluorescence, indicating preferential inhibition of efflux over uptake. In the presence of 10 microM digoxin (reduced efflux, enhanced cellular accumulation), cellular FL-MTX accumulation was specific, concentrative, and Na dependent. Thus transepithelial FL-MTX transport involved the following two carrier-mediated steps: electroneutral, Na-dependent uptake at the apical membrane and electroneutral efflux at the basolateral membrane. Finally, FL-MTX accumulation in both tissue compartments was reduced by phorbol ester and increased by forskolin, indicating antagonistic modulation by protein kinase C and protein kinase A.     

Human choroid plexus papilloma cells efficiently transport glucose and vitamin C

In vitro and in vivo studies suggest that the basolateral membrane of choroid plexus cells, which is in contact with blood vessels, is involved in the uptake of the reduced form of vitamin C, ascorbic acid (AA), through the sodium‐vitamin C cotransporter, (SVCT2). Moreover, very low levels of vitamin C were observed in the brains of SVCT2‐null mice. The oxidized form of vitamin C, dehydroascorbic acid (DHA), is incorporated through the facilitative glucose transporters (GLUTs). In this study, the contribution of SVCT2 and GLUT1 to vitamin C uptake in human choroid plexus papilloma (HCPP) cells in culture was examined. Both the functional activity and the kinetic parameters of GLUT1 and SVCT2 in cells isolated from HCPP were observed. Finally, DHA uptake by GLUT1 in choroid plexus cells was assessed in the presence of phorbol‐12‐myristate‐13‐acetate (PMA)‐activated human neutrophils. A marked increase in vitamin C uptake by choroid plexus cells was observed that was associated with superoxide generation and vitamin C oxidation (bystander effect). Thus, vitamin C can be incorporated by epithelial choroid plexus papilloma cells using the basolateral polarization of SVCT2 and GLUT1. This mechanism may be amplified with neutrophil infiltration (inflammation) of choroid plexus tumors.

     

Dichotomous development of the organic anion transport protein in liver and choroid plexus

Both adult liver and choroid plexus express the organic aniontransport protein (oatp1) and transport[³⁵S]bromosulfophthalein(BSP). Studies of the developing rat liver reveal that oatp1 mRNA andprotein do not begin to be expressed until 15 days postnatal and are atadult levels by 30 days. Uptake of[³⁵S]BSP follows thesame time course. In contrast, neonatal rat choroid plexus expressesoatp1 mRNA and protein. When quantified on a weight basis, the uptakeof [³⁵S]BSP in choroidplexus is lower in the adult than at earlier stages of development.Although fluorescence confocal microscopy of adult rat choroid plexusshows that oatp is localized to the apical surface, facing thecerebrospinal fluid, this method reveals an intracellular localizationof oatp1 in the neonate. Approximately 12 wk are required for theappearance of the adult pattern of distribution. Changes in thelocalization and activity of oatp1 during development could play animportant role in the pathobiology of maturation of the liver and thecentral nervous system.

     

Specificity and sodium dependence of the active nucleoside transport system in choroid plexus

The transport of [3H]deoxyuridine by the active nucleoside transport system into the isolated rabbit choroid plexus was measured in vitro under various conditions. Choroid plexuses were incubated in artificial CSF containing 1 microM [3H]deoxyuridine and 1 microM nitrobenzylthioinosine for 5 min under 95% O2-5% CO2 at 37 degrees C and the accumulation of [3H]deoxyuridine measured. Nitrobenzylthioinosine was added to the artificial CSF at a concentration (1 microM) that did not inhibit the active nucleoside transport system but did inhibit the separate, saturable nucleoside efflux system. The active transport of deoxyuridine into the choroid plexus depended on Na+ in the medium, as ouabain, substitution of Li+ and choline for Na+, and poly-L-lysine all inhibited deoxyuridine transport. Thiocyanate in place of chloride and penetrating sulfhydryl reagents also inhibited the active transport of deoxyuridine into choroid plexus. The active transport of deoxyuridine into choroid plexus, which is inhibited by naturally occurring ribo- and deoxyribonucleosides (IC50 = 7-21 microM), was not inhibited (IC50 much greater than 150 microM) by nucleosides with certain alterations on the 2', 3', or 5' positions in D-ribose or 2-deoxy-D-ribose (e.g., adenine arabinoside, 3'-deoxyadenosine, xylosyladenosine); or the pyrimidine or purine rings (e.g., 6-azauridine, xanthosine, 7-methylinosine, or 8-bromoadenosine). Other analogues were effective (IC50 = 8-26 microM; e.g., 5-substituted pyrimidine nucleosides, 7-deazaadenosine, 6-mercaptoguanosine) or less effective (IC50 = 46-145 microM; e.g., 5-azacytidine, 3-deazauridine) inhibitors of deoxyuridine transport into the isolated choroid plexus.     

Active transport of riboflavin by the isolated choroid plexus in vitro.

In vitro, the transport of [14C]riboflavin into and from the isolated choroid plexus, the anatomical locus of the blood-cerebrospinal fluid barrier, was studied. With concentrations of [14C]riboflavin of 0.7 microM (or greater) in the incubation medium, the choroid plexus accumulated [14C]riboflavin against a large concentration gradient by a process that did not depend on binding or intracellular metabolism of the [14C]riboflavin. The [14C]riboflavin accumulation process in isolated choroid plexus could be described by Michaelis-Menten transport kinetics (kt = 78 microM and Ymax = 1.65 mmol kg-1 (15 min)-1) and was inhibited by other flavins and probenecid but not by ribose, weak bases, or other B vitamins. The accumulation process was markedly depressed by iodoacetate and low temperatures. With a concentration of 0.08 microM [14C]riboflavin in the incubation medium, 28% of the [14C]riboflavin within the choroid plexus was converted to [14C]FAD or [14C]FMN intracellularly. Unlike the active transport of [14C]riboflavin into choroid plexus, accumulated [14C]riboflavin departed choroid plexus by a process independent of intracellular concentration or temperature. The efflux of [14C]riboflavin from choroid plexus could be described by first oder kinetics with a rate constant of -0.08 min-1.     

Molecular and kinetic parameters of sugar transport across the frog choroid plexus

Summary The nature of sugar transport across the blood-cerebrospinal fluid barrier has been investigated using anin vitro preparation of the frog posterior choroid plexus. The permeability of 41 sugars and related compounds was measured by the rapid osmotic procedure described previously. Sugar permeation was found to be stereospecific, inhibited by 1,5-difluoro-2,4-dinitrobenzene, insensitive to anoxia, and independent of the external alkali cation composition. In addition, the transport of a sugar was inhibited by structural analogues. Transport occurred equally well from the ventricular or serosal surface of the tissue, and the rate of transport could be described formally by Michaelis-Menten kinetics. The results were analyzed in terms of the conformation of the sugars in aqueous solution. Sugars which were transported have the d-glucose chair conformation. There is a good correlation between the affinity of the sugar for the transport system and the number of hydroxyl groups attached to the equatorial plane of the ring; d-glucose with five equatorial hydroxyl groups has the greatest affinity. It is concluded that sugar transport across the choroid plexus occurs by facilitated diffusion.