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.     

VITAMIN B6 TRANSPORT IN THE CENTRAL NERVOUS SYSTEM: IN VITRO STUDIES

Abstract— The transport into and release of tritium labeled vitamin B₆ ([³H]B₆) from rabbit brain slices and isolated choroid plexuses were studied. In vitro, both brain slices and choroid plexus concentrated [³H]B₆ by an energy dependent uptake system when [³H]pyridoxine (PIN) was added to the incubation medium. Most of the [³H] within the tissues was phosphorylated [³H]B₆. In each tissue, the nonphosphorylated vitamers inhibited the uptake of [³H]PIN from the medium significantly more than the phosphorylated vitamers. The concentrations of the nonphosphorylated B₆ vitamers necessary to inhibit brain and choroid plexus uptake of [³H]PIN from the medium by 50% were approx 0.4 μm and 5–10μm respectively after a 30 min incubation. Both brain slices and choroid plexus readily released (46 and 56% respectively in 30 min) previously accumulated [³H]B₆ into artificial CSF. However, brain slices released only nonphosphorylated [³H]B₆, whereas the choroid plexus released predominantly phosphorylated [³H]B₆. Addition of unlabeled PIN to the release media significantly increased the percentage of [³H]B₆ released by both brain slices and choroid plexus. The results of these in vitro studies provide evidence that: (1) both brain slices and chloroid plexus possess specific uptake and release mechanisms for B₆, and (2) these mechanisms tend to regulate intracellular B₆ levels. These studies also suggest that the choroid plexus serves as a locus for the transfer of B₆ from blood to CSF and is the source of most of the phosphorylated B₆ in CSF.     

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.     

NIACIN AND NIACINAMIDE ACCUMULATION BY RABBIT BRAIN SLICES AND CHOROID PLEXUS IN VITRO

The transport into and release of¹⁴C-labeled niacin and niacinamide from rabbit brain slices and isolated choroid plexuses were studied. In vitro, both brain slices and choroid plexus concentrated ¹⁴C by specific, energy-dependent mechanisms when [¹⁴C]niacinamide was added to the incubation medium. The saturable accumulation velocities, which were linear for 30 min, depended, in part, on incorporation of the [¹⁴C]niacinamide into NAD. The X_T and Y_max for ¹⁴C accumulation with [¹⁴C]niacinamide in the medium by brain slices and choroid plexus were 0.80 μM and 1.45 μmolkg^?1 (30 min)^?1, and 0.23 μM and 18.6 μmol kg^?1 (30 min)^?1 respectively. In vitro, the choroid plexus, unlike brain slices, vigorously concentrated ¹⁴C by a separate, specific energy-dependent process when ¹⁴C niacin was added to the incubation medium. The saturable accumulation velocity, which was linear for 30 min, depended completely on the metabolism of [¹⁴C]niacin. The K_T and Y_max for¹⁴C accumulation by choroid plexus with [¹⁴C]niacin in the medium were 18.1 μM and 439 μmol kg^?1 (30 min)^?1 respectively. Whether preincubated in [¹⁴C]niacin or [¹⁴C]niacinamide, choroid plexus released predominantly [¹⁴C]niacinamide.     

The movement of sulfate and thiosulfate into in vivo choroid plexus

The accumulation of sulfate (SO₄?) and thiosulfate (S₂O₃?) in the choroid plexus, brain, and cerebrospinal fluid (CSF) of the rat was measured at various plasma levels of these anions. Increasing the plasma SO₄ ? or S₂O₃ ? concentration levels 40- and 580-fold, respectively, resulted in a linear increase in CSF, brain and choroid plexus concentration of these ions. The relationship between the concentration of these ions in CSF and choroid plexus was also approximately linear over a wide CSF concentration range. In addition, S₂O₃? did not appear to influence the relation between the concentration of SO₄? in choroid plexus and CSF. The results seem to indicate that there is no saturation of the mechanism responsible for maintaining the low SO₄? or S₂O₃? concentration in CSF nor does there appear to be competition between these anions for clearance from the CSF. These findings are in conflict with data supporting the active transport of SO₄? and S₂O₃? from the CSF across the CSF-blood barrier (choroid plexus).     

The permeability of the frog choroid plexus to nonelectrolytes

Summary Anin vitro preparation of the frog choroid plexus has been used to measure the permeability of the choroidal epithelium to 50 nonelectrolytes by an osmotic method. The method involves the measurement of nonelectrolyte reflection coefficients () by a rapid electrical procedure. For the majority of compounds tested, there was a good correlation between the rate of solute permeation and the solute's bulk-phase lipid: water partition coefficients; i.e., the higher the partition coefficient the greater the permeability. The membrane lipids of the choroid plexus differ from the membrane lipids of the gall bladder in at least three ways: (1) the lipids of the choroid plexus cannot distinguish between branched chain solutes and their straight chain isomers; (2) small polar solutes such as urea and acetamide permeate via the membrane lipids to a significant extent; and (3) the smaller selectivity ratios suggest that the lipids of the choroid plexus contain more hydrogen bonding sites (i.e., there are stronger solute: lipid intermolecular forces in the choroid plexus). The permeability characteristics of the choroid plexus are qualitatively similar to those of most other cell membranes. In addition, there is evidence for the presence of a special mechanism for the transport of sugar across this epithelium.     

THE SPECIFICITY AND SULFHYDRYL SENSITIVITY OF THE INOSITOL TRANSPORT SYSTEM OF THE CENTRAL NERVOUS SYSTEM

Abstract— The transport of two cyclohexitol stereoisomers, myo-inositol (inositol) and scyllo-inositol (scyllitol), from blood into the CNS in vivo and into the choroid plexus in vitro was studied. In vitro , the uptake of [³H]scyllitol or [³H]inositol by choroid plexuses, isolated from rabbits and incubated in artificial CSF, was measured. Both scyllitol and inositol inhibited [³H]scyllitol or [³H]inositol accumulation by the choroid plexus. Inositol competitively inhibited [³H]scyllitol accumulation and both isomers had a comparable affinity (K_t= 0.1 m m ) for the single cyclohexitol accumulation system. The other 6 stereoisomers tested had an order of magnitude less affinity for the cyclohexitol accumulation system of choroid plexus. Thiol reagents that penetrate cells inhibited inositol accumulation by choroid plexus more effectively than nonpenetrating thiol reagents. In vivo , in unanesthetized rabbits. the transport of unmetabolized [³H]inositol from blood into CSF, choroid plexus and brain was readily saturated by increasing the plasma levels of myo-inositol but not by the stereoisomer d -chiroinositol. Similarly, the transport of unmetabolized [³H]scyllitol into CSF, brain and choroid plexus was readily saturated by increasing the plasma levels of myo-inositol. Beside documenting the stereospecificity and thiol reagent sensitivity of the inositol transport mechanism of the choroid plexus, these results provide further evidence that the choroid plexus is a locus for cyclohexitol transport between blood and CSF. Moreover, they show that scyllitol, which, like inositol, is present at a higher concentration in brain than plasma, can be transported from blood into CSF and brain by the same system that transports inositol.     

Serotonin 5-HT2C Receptor Stimulates Cyclic GMP Formation in Choroid Plexus

Abstract: The serotonin 5-HT_2C receptor (formerly designated the 5-HT_1C receptor) of the choroid plexus triggers phosphoinositide turnover. In the present study, we demonstrate that receptor activation also triggers the formation of cyclic GMP (cGMP). Application of 1 µM 5-HT to porcine choroid plexus tissue slices resulted in stimulation of cGMP formation to a maximum of five-fold basal level, with an EC₅₀ of 11 nM. This response was not inhibited by muscarinic or β-adrenergic receptor antagonists. Serotonin receptor antagonists inhibited cGMP formation with apparent K_i values of 1.3 (mianserin), 200 (ketanserin), and 5,500 (spiperone) nM, respectively. Neither serotonin-stimulated cGMP formation nor PI turnover was inhibited by pertussis toxin pretreatment. Preliminary biochemical studies suggested that serotonin-stimulated cGMP formation was calcium, phospholipase A₂, and lipoxygenase dependent, as incubation in low calcium buffers or inclusion of the phospholipase A₂ or lipoxygenase inhibitors p-bromophenacyl bromide or BW 755c resulted in significant reduction of cGMP formation. The present results suggest that in addition to triggering phosphoinositide turnover, choroid plexus serotonin 5-HT_2C receptors trigger cGMP formation in a calcium-sensitive manner.     

Role of the blood–cerebrospinal fluid barrier transporter as a cerebral clearance system for prostaglandin E2 produced in the brain

An increasing level of prostaglandin (PG) E₂ is involved in the progression of neuroinflammation induced by ischemia and bacterial infection. Although an imbalance in the rates of production and clearance of PGE₂ under these pathological conditions appears to affect the concentration of PGE₂ in the cerebrospinal fluid (CSF), the regulatory system remains incompletely understood. The purpose of this study was to investigate the cellular system of PGE₂ production via microsomal PGE synthetase‐1 (mPGES‐1), the inducible PGE₂‐generating enzyme, and PGE₂ elimination from the CSF via the blood–CSF barrier (BCSFB). Immunohistochemical analysis revealed that mPGES‐1 was expressed in the soma and perivascular sheets of astrocytes, pia mater, and brain blood vessel endothelial cells, suggesting that these cells are local production sites of PGE₂ in the CSF. The in vivo PGE₂ elimination clearance from the CSF was eightfold greater than that of d ‐mannitol, which is considered to reflect CSF bulk flow. This process was inhibited by the simultaneous injection of unlabeled PGE₂ and β‐lactam antibiotics, such as benzylpenicillin, cefazolin, and ceftriaxone, which are substrates and/or inhibitors of organic anion transporter 3 (OAT3). The characteristics of PGE₂ uptake by the isolated choroid plexus were at least partially consistent with those of OAT3. OAT3 was able to mediate PGE₂ transport with a Michaelis–Menten constant of 4.24 μM. These findings indicate that a system regulating the PGE₂ level in the CSF involves OAT3‐mediated PGE₂ uptake by choroid plexus epithelial cells, acting as a cerebral clearance pathway via the BCSFB of locally produced PGE₂.     

Altered pH(i) regulation and Na(+)/HCO3(-) transporter activity in choroid plexus of cilia-defective Tg737(orpk) mutant mouse

Tg737^orpk mice have defects in cilia assembly and develop hydrocephalus in the perinatal period of life. Hydrocephalus is progressive and is thought to be initiated by abnormal ion and water transport across the choroid plexus epithelium. The pathology is further aggravated by the slow and disorganized beating of motile cilia on ependymal cells that contribute to decreased cerebrospinal fluid movement through the ventricles. Previously, we demonstrated that the hydrocephalus phenotype is associated with a marked increase in intracellular cAMP levels in choroid plexus epithelium, which is known to have regulatory effects on ion and fluid movement in many secretory epithelia. To evaluate whether the hydrocephalus in Tg737^orpk mutants is associated with defects in ion transport, we compared the steady-state pH_i and Na⁺-dependent transport activities of isolated choroid plexus epithelium tissue from Tg737^orpk mutant and wild-type mice. The data indicate that Tg737^orpk mutant choroid plexus epithelium have lower pH_i and higher Na⁺-dependent HCO₃^– transport activity compared with wild-type choroid plexus epithelium. In addition, wild-type choroid plexus epithelium could be converted to a mutant phenotype with regard to the activity of Na⁺-dependent HCO₃^– transport by addition of dibutyryl-cAMP and mutant choroid plexus epithelium toward the wild-type phenotype by inhibiting PKA activity with H-89. Together, these data suggest that cilia have an important role in regulating normal physiology of choroid plexus epithelium and that ciliary dysfunction in Tg737^orpk mutants disrupts a signaling pathway leading to elevated intracellular cAMP levels and aberrant regulation of pH_i and ion transport activity. cAMP; ion transport     

Choroid epithelial cell pH.

The aim of the study was to obtain a reliable estimate of the pH of the parenchymal ($\text{i.e.}$, epithelial) cell compartment of the choroid plexus. From the analysis of the distribution of ¹⁴C-dimethyloxazol-idinedione (DMO) and ³H-inulin in the lateral ventricular plexus, a value of 7.0 can be calculated for choroid cell pH. Because of the presence in choroid plexus of carrier-transport systems for various organic acids, the effect of carrier DMO on the measurement of cell pH was ascertained. The addition of carrier DMO (15 mg/kg, i.p.) did not significantly affect the value of choroid cell pH as calculated from the steady-state distribution of ¹⁴C-DMO. Thus if DMO is actively transported by the choroid plexus, it is probably translocated by a system that is saturated by low concentrations of carrier (ca 1 mg/kg, i.p.).     

ENZYMES OF THE γ-GLUTAMYL CYCLE IN THE CHOROID PLEXUS AND BRAIN

—The presence of enzymes of the γ-glutamyl cycle in the bovine and rabbit brain and choroid plexus is described. The activities of γ-glutamyl transpeptidase, γ-glutamyl cyclotransferase and γ-glutamyl-cysteine synthetase in the choroid plexus were found to be higher than in the brain. The activity of γ-glutamyl transpeptidase in the choroid plexus was many times higher than the activity of the other enzymes. Brain and choroid plexus γ-glutamyl transpeptidase were activated by Na⁺ and K⁺. Both brain and choroid plexus showed only a very limited capacity to metabolize [¹⁴C]5-oxoproline to ¹⁴CO₂.     

NBCe2 exhibits a 3 HCO3(-):1 Na+ stoichiometry in mouse choroid plexus epithelial cells

The transport stoichiometry of the electrogenic sodium-bicarbonate cotransporter (SLC4A5 or NBCe2) in mouse choroid plexus was examined. Whole-cell recording methods measured the currents carried by the NBCe2, using experimental solutions determined to minimise the contributions of the other ion conductances present. Increases in outward current were observed when 21.2 mM was added to the bath solution in the presence of Na⁺, but not N-methyl-d-glucamine. This -induced current was completely abolished by 500 μM 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid. The reversal potential for the -induced current was −95.1 ± 7.1 mV (n = 11), a value which corresponds to a NBCe2 transport stoichiometry of 3 with 1 Na⁺. The NBCe2, with this stoichiometry, will mediate the efflux of and Na⁺ from the cell into the cerebrospinal fluid at the apical membrane of the choroid plexus.     

Calcium-dependent accumulation induced by carbamylcholine of guanosine 3′,5′-monophosphate in rabbit choroid plexus

An active guanylate cyclase system was detected in isolated choroid plexus of rabbits by sodium azide (6 × 10^?5 mol/l) which increased cGMP levels tenfold within 15 min. Inhibition of cGMP phosphodiesterase by sodium azide was excluded. cGMP accumulation was also raised dose-dependently by carbamylcholine, a cholinergic agonist. Pretreatment of chroid plexus with atropine (10^?7 mol/l) reduced the effect of carbamylcholine (5 × 10^?5 mol/l) by 80%. Both carbamylcholine and sodium azide induced accumulation of cGMP also in the incubation medium, indicating rapid extrusion of the nucleotide from choroid plexus cells. The effect of carbamylcholine could be mimicked by the calcium ionophore A 23187. Incubation in calcium-free medium abolished cGMP accumulation by carbamylcholine and A 23187 but not by sodium azide, indicating a different mechanism of action. Sodium azide, carbamylcholine and A 23187 had no effect on cyclic AMP levels. Withdrawal of calcium led to an enhanced efflux of both cAMP and cGMP. Since a cholinergic innervation of stroma and epithelial cells has been described, we hypothesize that cGMP and calcium may be involved in cholinergic transmission regulating blood flow or transport processes of the 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.     

In vitro transcriptome analysis of porcine choroid plexus epithelial cells in response to Streptococcus suis: release of pro-inflammatory cytokines and chemokines

The Gram-positive zoonotic bacterium Streptococcus suis (S. suis) is responsible for a wide range of diseases including meningitis in pigs and humans. The blood-cerebrospinal fluid (CSF) barrier is constituted by the epithelial cells of the choroid plexus, which execute barrier function also after bacteria have entered the central nervous system (CNS). We show that the bacterial capsule, a major virulence factor, strongly attenuates adhesion of S. suis to the apical side of porcine choroid plexus epithelial cells (PCPEC). Oligonucleotide microarray analysis and quantitative PCR surprisingly demonstrated that adherent wild-type and capsule-deficient S. suis influenced expression of a pronounced similar pattern of genes in PCPEC. Investigation of purified capsular material provided no evidence for a significant role of the capsule. Enriched among the regulated genes were those involved in “inflammatory response”, “defense response” and “cytokine activity”. These comprised several cytokines and chemokines including the interleukins 6 and 8, which could be detected on protein level. We show that after infection with S. suis the choroid plexus contributes to the immune response by actively producing cytokines and chemokines. Other virulence factors than the bacterial capsule may be relevant in inducing a strong inflammatory response in the CNS during S. suis meningitis.     

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     

Fine Structure and Histochemistry of the Choroid Plexus of the Teleost Leuciscus rutilus

Analyses of the fine structure of the posterior choroid plexus in the teleost Leuciscus rutilus and the determination of the presence and function of the enzymes acid and alkaline phosphatases, ATPase and glucose-6-phosphatase confirm similarities between these epithelial cells and the saccus dorsalis and also with the epithelial cells of the choroid plexus found in mammals. The teleost plexus cells contain coated vesicles which are derived from the plasmalemma as well as from the Golgi complex. Moreover, they contain multivesicular bodies and Iysosomes. These organelles function in the absorption of substances from the cerebrospinal fluid and in the breakdown of these substances within the cells. The investigated enzymes play an important role in the secretion of electrolytes into the cerebrospinal fluid by active membrane transport.     

The Vascularization of the Anuran Brain Diencephalon and Choroid Plexus: A scanning electron microscopical study of vascular corrosion casts

Albrecht, U., Lametschwandtner, A., Adam, H. 1979. The vascularization of the anuran brain. Diencephalon and choroid plexus. A scanning electron microscopical study of vascular corrosion casts. (Department of Zoology, University of Saulzburg, Austria.) — Acta zool. (Stockh.) 61(4): 203–220. The vascularization of the diencephalon (with choroid plexus of the third ventricle, epithalamus and pineal region, thalamus and hypothalamus) of the toad, Bufo bufo (L.) has been studied by means of scanning electron microscopy of vascular corrosion casts. To localize angioarchitectonic patterns of distinct diencephalic regions the authors refer to critical point dried specimens and to histological sections. In the choroid plexus a supply via one choroid artery, which arises from the posterior telencephalic artery, was found. Its strict dichotomous branching is pointed out. In generally a similar vascular pattern like that in the choroid plexus of the fourth ventricle has to be reported. Furthermore the epithalamic region with the epiphysial area was under investigation. No special angioarchitecture of the epiphysis was found. There are also no prominent vascular connections with thalamic or hypothalamic regions. The thalamic region is supplied by branches of the posterior telencephalic artery as well as by branches of the preoptic artery. Epithalamic and thalamic regions are drained via the posterior diencephalic vein. Special attention was also paid to the preoptic, the chiasmatic and the retrochiasmatic area. No special vascular connections, however, were found.