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.     

Cystic Fibrosis Transmembrane Conductance Regulator Is Found Within Brain Ventricular Epithelium and Choroid Plexus

Abstract: The cystic fibrosis gene product, cystic fibrosis transmembrane conductance regulator (CFTR), functions as a CI⁻ channel that is regulated by cyclic AMP-dependent phosphorylation. We have investigated the expression of CFTR protein in the rodent brain by both western blotting of samples prepared by microdissection and immunohistochemistry. CFTR was found to be expressed in choroid plexus and ependyma. In tissue sections, CFTR-like immunoreactivity was concentrated in fine puncta localized about 1–2 µm from the CSF-contacting side of ependyma and choroid plexus. CFTR in choroid plexus may play a role in the regulation of the composition of CSF by cyclic AMP-elevating agents, but the role of this chloride transporter in ependymal function remains to be determined.     

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.     

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.     

Expression of aquaporin 1 and aquaporin 4 water channels in rat choroid plexus

The role of aquaporins in cerebrospinal fluid (CSF) secretion was investigated in this study. Western analysis and immunocytochemistry were used to examine the expression of aquaporin 1 (AQP1) and aquaporin 4 (AQP4) in the rat choroid plexus epithelium. Western analyses were performed on a membrane fraction that was enriched in Na⁺/K⁺-ATPase and AE2, marker proteins for the apical and basolateral membranes of the choroid plexus epithelium, respectively. The AQP1 antibody detected peptides with molecular masses of 27 and 32 kDa in fourth and lateral ventricle choroid plexus. A single peptide of 29 kDa was identified by the AQP4 antibody in fourth and lateral ventricle choroid plexus. Immunocytochemistry demonstrated that AQP1 is expressed in the apical membrane of both lateral and fourth ventricle choroid plexus epithelial cells. The immunofluorescence signal with the AQP4 antibody was diffusely distributed throughout the cytoplasm, and there was no evidence for AQP4 expression in either the apical or basolateral membrane of the epithelial cells. The data suggest that AQP1 contributes to water transport across the apical membrane of the choroid plexus epithelium during CSF secretion. The route by which water crosses the basolateral membrane, however, remains to be determined.     

Immunohistochemical study of localization of gamma-glutamyl transpeptidase in the rat brain

Gamma-glutamyl transpeptidase (gamma-GTP) is a membrane-bound enzyme which is known to play a crucial role in active transport of amino acids across membrane barriers. We prepared a monoclonal antibody recognizing specifically rat gamma-GTP and investigated localization of the enzyme in the rat brain by immunohistochemistry with this antibody. The antigen was localized on the ependyma, epithelia of the choroid plexus and microvessels. More precise localization of gamma-GTP was examined with immuno-electron microscopy. The antigen was recognized on the microvilli and cilia of the ependymal cells, microvilli of the choroid epithelial cells and luminal membranes of the vascular endothelial cells.     

In situ localization of transthyretin-mRNA in the adult human liver,choroid plexus and pancreatic islets and in endocrine tumours of the pancreas and gut

Summary In situ hybridization with ³⁵S-labeled single stranded RNA probes was used on sections from formaldehyde-fixed and paraffin-embedded tissue specimens to provide semiquantitative data on the occurrence of transthyretin(TTR)-mRNA in human liver, choroid plexus and pancreatic islets as well as in 15 endocrine tumours of the pancreas and gut. A monoclonal antibody to TTR was used for immunocytochemical identification of the protein in consecutive sections.The amount of TTR-mRNA in hepatocytes was found to be much less than that in epithelial cells of the choroid plexus. Glucagon cells of the pancreatic islets were also specifically labeled and the level of TTR-mRNA in these cells was intermediate between that of hepatocytes and choroid plexus epithelial cells. Four glucagonomas, one malignant insulinoma and two midgut carcinoids were shown to contain TTR-mRNA. The in situ labeled cells were also found to be TTR immunoreactive. These findings present the first conclusive evidence for TTR synthesis in pancreatic islets and in endocrine tumours. They also establish that the high serum concentration of TTR found in some patients with endocrine tumours (notably glucagonomas) is most likely due to tumour production of TTR.     

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.     

Nucleoside Transporter of Cerebral Micro vessels and Choroid Plexus

The nucleoside transporter of cerebral microvessels and choroid plexus was identified and characterized using [3H]nitrobenzylthioinosine (NBMPR) as a specific probe. [3H]NBMPR bound reversibly and with high affinity to a single specific site in particulate fractions of cerebral microvessels, choroid plexus, and cerebral cortex of the rat and the pig. The dissociation constants (KD 0.1-0.7 nM) were similar in the various tissue preparations from each species, but the maximal binding capacities (Bmax) were about fivefold higher in cerebral microvessels and choroid plexus than in the cerebral cortex. Nitrobenzylthioguanosine and dipyridamole were the most potent competitors for [3H]NBMPR binding. Several naturally occurring nucleosides displaced specific [3H]NBMPR binding to cerebral microvessels in vitro, in a rank order that correlated well with their ability to cross the blood-brain barrier in vivo. Adenosine analogues and theophylline were less effective in displacing [3H]NBMPR binding than in displacing adenosine receptor ligands. Photoactivation of cerebral microvessels and choroid plexus bound with [3H]NBMPR followed by solubilization and polyacrylamide gel electrophoresis labeled a protein(s) with a molecular weight of approximately 60,000. These results indicate that cerebral microvessels and choroid plexus have a much higher density of the nucleoside transporter moiety than the cerebral cortex and that this nucleoside transporter has pharmacological properties and a molecular weight similar to those of erythrocytes and other mammalian tissues.     

Arginine vasopressin causes morphological changes suggestive of fluid transport in rat choroid plexus epithelium

Summary The experiments described herein use an in vitro preparation of choroid plexus to demonstrate that it is a vasopressin-responsive organ by morphologic criteria. Choroid plexus from rats was incubated for one hour in graded concentrations of arginine vasopressin (AVP). Within physiologic range of molar concentration, incubation in vasopressin induced a decrease in basal and lateral spaces in choroid plexus epithelial cells as well as an increase in number of dark cells. The number of cells with basal spaces decreased significantly from 82.7±9.2 in control tissue to 19±18 in tissue incubated in 10^-12 M AVP; similarly, the number with lateral cellular spaces decreased from 20±8.8 to 7.6±2.2 cells in 10^-10 M AVP. Dark cells increased in number from 3.8±2.6 in control conditions to 49±4 with 10^-9 M vasopressin. These data suggest important effects of arginine vasopressin in cerebrospinal fluid (CSF) on choroid plexus, compatible with enhanced fluid transport across choroid epithelial cells.     

Histochemistry of proteases in ependyma,choroid plexus and leptomeninges

Summary Aminopeptidase M (APM), aminopeptidase A (APA), dipeptidyl peptidase IV (DPP IV) and -glutamyl transferase (GGT) were demonstrated histochemically in cryostat sections of the rat brain to show the reaction pattern of ependyma, choroid plexus and leptomeninges. GGT was only demonstrable in the cell membranes of ependymal cells and in the leptomeninges; however, APA, APM and DAP IV showed a variable degree of activity in the capillary endothelium of the choroid plexus as well as in the leptomeninges. On the basis of these results, it is postulated that peptides in the cerebrospinal fluid can be cleaved extraventricularly by the enzymes demonstrated in the leptomeninges.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Development of transport mechanisms for sulphate and iodide in immature choroid plexus

The time-course of development of sulphate and iodide transport mechanisms in choroid plexus was studied by measuring uptake of [³⁵S]sulphate and [¹²⁵I]iodide from an incubating medium by isolated choroid plexuses of foetal and newborn rabbits and cats. Sulphate uptake by choroid plexus was poorly developed in rabbit foetuses just before term, but highly developed in newborn animals. Iodide uptake was already well developed in the most immature foetuses studied.     

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.     

Glutaric aciduria type I and methylmalonic aciduria: Simulation of cerebral import and export of accumulating neurotoxic dicarboxylic acids in in vitro models of the blood–brain barrier and the choroid plexus

Intracerebral accumulation of neurotoxic dicarboxylic acids (DCAs) plays an important pathophysiological role in glutaric aciduria type I and methylmalonic aciduria. Therefore, we investigated the transport characteristics of accumulating DCAs – glutaric (GA), 3-hydroxyglutaric (3-OH-GA) and methylmalonic acid (MMA) – across porcine brain capillary endothelial cells (pBCEC) and human choroid plexus epithelial cells (hCPEC) representing in vitro models of the blood–brain barrier (BBB) and the choroid plexus respectively. We identified expression of organic acid transporters 1 (OAT1) and 3 (OAT3) in pBCEC on mRNA and protein level. For DCAs tested, transport from the basolateral to the apical site (i.e. efflux) was higher than influx. Efflux transport of GA, 3-OH-GA, and MMA across pBCEC was Na⁺-dependent, ATP-independent, and was inhibited by the OAT substrates para-aminohippuric acid (PAH), estrone sulfate, and taurocholate, and the OAT inhibitor probenecid. Members of the ATP-binding cassette transporter family or the organic anion transporting polypeptide family, namely MRP2, P-gp, BCRP, and OATP1B3, did not mediate transport of GA, 3-OH-GA or MMA confirming the specificity of efflux transport via OATs. In hCPEC, cellular import of GA was dependent on Na⁺-gradient, inhibited by NaCN, and unaffected by probenecid suggesting a Na⁺-dependent DCA transporter. Specific transport of GA across hCPEC, however, was not found. In conclusion, our results indicate a low but specific efflux transport for GA, 3-OH-GA, and MMA across pBCEC, an in vitro model of the BBB, via OAT1 and OAT3 but not across hCPEC, an in vitro model of the choroid plexus.     

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.     

Deoxycytidine Transport and Metabolism in the Central Nervous System

Abstract: The mechanisms by which deoxycytidine enters and leaves brain, choroid plexus, and CSF were investigated by injecting [³H]deoxycytidine intraarterially, intravenously, and intraventricularly. After intracarotid injection of deoxycytidine (1.0 μM) into rats, deoxycytidine did not pass through the blood-brain barrier at a faster rate than sucrose. [³H]Deoxycytidine, either alone or together with unlabeled deoxycytidine, was infused at a constant rate into conscious adult rabbits. At 130 min, [³H]deoxycytidine readily entered CSF, choroid plexus, and brain. In brain, approx. 60% of the nonvolatile radioactivity was attributable to [³H]deoxycytidine phosphates. The addition of 0.22 mmol/kg unlabeled deoxycytidine to the infusion syringe decreased the phosphorylation of [³H]deoxycytidine in brain by approx. 50%; the addition of 2.2 mmol/kg of unlabeled deoxycytidine to the infusion syringe decreased the relative entry of [³H]deoxycytidine into CSF and brain by approx. 50 and 75%, respectively. Two hours after the intraventricular injection of [³H]deoxycytidine, [³H]deoxycytidine was rapidly cleared from CSF, in part, to brain, where approx. 65% of the [³H]deoxycytidine was converted to [³H]deoxycytidine phosphates. The intraventricular injection of unlabeled deoxycytidine with the [³H]deoxycytidine decreased the phosphorylation of [³H]deoxycytidine in the brain significantly and also decreased the clearance of [³H]deoxycytidine from the CSF. These results were interpreted as showing that the entry of deoxycytidine from blood into CSF occurs by a saturable transport system within the choroid plexus. Once within the CSF, the deoxycytidine can enter brain, undergo phosphorylation to deoxycytidine phosphates, and subsequently be incorporated into DNA.     

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.     

Enzymatic organization of the subcommissural organ.

In the subcommissural organ (SCO) of the guinea pig, rat, golden hamster, and mouse the activity and distribution of enzymes related to the energy-supplying metabolism and of some marker enzymes of different cell organelles have been investigated by means of mostly modified histochemical methods. The results were compared with findings in the ciliated ependyma of the ventricular wall and with those in the ependyma of the choroid plexus of the third ventricle. In the ependymal part of the SCO only a moderate activity of hexokinase is observed in its specialized columnar cells whereas a high activity is present both in the ciliated ependyma and the choroid plexus. - The staining pattern of glucose-6-phosphatase is similar to that of hexokinase but this enzyme is found is the SCO only. - Likewise hexokinase, glycogen granules and enzymes related to glycogen metabolism (phosphoglucomutase, uridine-diphosphoglucose pyrophosphorylase, glycogen synthetase and phosphorylase) are regularly found most numerous and active in the nuclear and supra-nuclear area of the ependymal part. These enzymes are less active in both the other ependymal regions. - Uridine-diphosphoglucose dehydrogenase could not be demonstrated in the SCO. The NADP-linked enzymes of the pentose phosphate shunt, glucose-6-phosphate and 6-phosphogluconate dehydrogenase, show a moderate activity which decreases also from the nuclear towards the apical area of the ependymal cells of the SCO. Enzymes of the glycolytic pathway, such as glucosephosphate isomerase, fructose-6-phosphate kinase, fructose-I,6-diphosphate aldolase, glyceraldehyde-3-phosphate and lactate dehydrogenase, are highly active in the SCO and are located mainly in the supranuclear area, too. Fructose-1,6-diphosphatase could not be demonstrated thus indicating that in the SCO the pathway is most probably only glycolytic but not gluconeogenetic. Compared to the ependyma of the ventricular wall and of the choroid plexus, in the SCO the M type subunits of lactate dehydrogenase predominate. Glycolytic enzymes are also very active in the choroid plexus but less in the ciliated ependyma. Compared to the ciliated ependyma and especially to the ependyma of the choroid plexus, the activities of enzymes which are only present in mitochondria (NAD-linked isocitrate dehydrogenase, succinate dehydrogenase, NAD-linked malate dehydrogenase after preextraction, cytochrome oxidase, 3-hydroxybutyrate and glycerolphosphate and glutamate dehydrogenase) are relatively low. Mitochondria are accumulated near the superior pole of the nuclei as well as in the most apical part of the ependymal cells. - The staining pattern of NADP-linked isocitrate and malate dehydrogenase as well as of NADH dehydrogenase suggests that these enzymes are localized both in and out of mitochondria. The extramitochondrial activity of the first two enzymes might be localized in the cytosol. The extramitochondrial activity of NADH dehydrogenase might be localized in the endoplasmic reticulum...     

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.     

An extracellular retinol-binding glycoprotein in the rat eye—characterization,localization and biosynthesis

We have identified and partially purified interstitial retinol-binding protein (IRBP) from the subretinal space of the rat. It appeared to be glycosylated. Its apparent mol. wt was 270,000 by gel-filtration and 144,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Rat IRBP cross-reacted with anti-bovine IRBP sheep and rabbit sera, bound all-trans-[15-³H] retinol and was bound by concanavalin A. IRBP was not detected in the cytosols of the neural retina or retinal pigment epithelium and choroid. This distribution was confirmed by immunocytochemistry using a fluorescence-labeled second antibody. Immunospecific fluorescence was most intense in the interphotoreceptor matrix in a 6.5 μm band adjacent to the retinal pigment epithelium. It was less intense over the remainder of the rod outer segment layer and was comparatively faint over the inner segment region. Its occurrence in the interstitial space between the photoreceptors and retinal pigment epithelium coupled with the fact it bound all-trans-[15-³H] retinol supports the concept that it may be implicated in the transport of retinoids between the retina and the retinal pigment epithelium during the visual cycle. When incubated with [³H]leucine or [³H]glucosamine, isolated retinas (but not retinal pigment epithelium and choroid) secreted labeled IRBP into the medium. This suggests that the retina plays a role in regulating the amount of IRBP in the subretinal space.