Rapamycin Treatment Reduces Brain Pericyte Constriction in Ischemic Stroke.

The contraction and subsequent death of brain pericytes may play a role in microvascular no-reflow following the reopening of an occluded artery during ischemic stroke. Mammalian target of rapamycin (mTOR) inhibition has been shown to reduce motility/contractility of various cancer cell lines and reduce neuronal cell death in stroke. However, the effects of mTOR inhibition on brain pericyte contraction and death during ischemia have not yet been investigated. Cultured pericytes exposed to simulated ischemia for 12 h in vitro contracted after less than 1 h, which was about 7 h prior to cell death. Rapamycin significantly reduced the rate of pericyte contraction during ischemia; however, it did not have a significant effect on pericyte viability at any time point. Rapamycin appeared to reduce pericyte contraction through a mechanism that is independent of changes in intracellular calcium. Using a mouse model of middle cerebral artery occlusion, we showed that rapamycin significantly increased the diameter of capillaries underneath pericytes and increased the number of open capillaries 30 min following recanalisation. Our findings suggest that rapamycin may be a useful adjuvant therapeutic to reduce pericyte contraction and improve cerebral reperfusion post-stroke.

Pre-aggregated Aß1-42 peptide increases tau aggregation and hyperphosphorylation after short-term application.

Neuritic amyloid plaques and neurofibrillary tangles, consisting of hyperphosphorylated tau protein, are the hallmarks of Alzheimer disease. It is not clear so far, how both structures are functionally and physiologically connected. We have investigated the role of Aß1-42 on hyperphosphorylation and aggregation of tau in SY5Y cells by transfection and overexpression with two tau constructs, a shortened wildtype tau (2N4R) and a point mutation tau (P301L), found in fronto-temporal dementia. It was found that the tau protein becomes hyperphosphorylated and forms large aggregates inside cells, visualized by immunofluorescence, after short incubation of 90 min with preaggregated Aß1-42. In Addition, Aß1-42 caused a decrease of tau solubility in both tau constructs in this relatively short time period. Taken together, these experiments suggest that pathological preaggregated Aß1-42 in physiological concentrations quickly induces hyperphosphorylation and pathological structural changes of tau protein and thereby directly linking the 'amyloid hypothesis' to tau pathology, observed in Alzheimer disease.

Effects of hypoxia, glucose deprivation and recovery on the expression of nucleoside transporters and adenosine uptake in primary culture of rat cortical astrocytes.

The aim of this study was to explore effects of hypoxia, glucose deprivation (HGD) and recovery on expression and activities of equilibrative nucleoside transporters (rENT) and concentrative nucleoside transporters (rCNT) in rat astrocytes in primary culture. Amounts of cellular ATP in the control group (CG, 5% CO(2) in air, medium containing 7 mM D-glucose, 1 mM Na(+)-pyruvate, 1 h), HGD group (2% O(2)/5% CO(2) in N(2), pyruvate-free medium containing 1.5 mM D-glucose and 10 mM 2-deoxy-D-glucose, 1 h) and recovery group (RG, HGD for 1 h, followed by 1 h exposure to the same conditions as the CG) were (nmol/mg protein, n = 4) 18 +/- 1.6, 4.9 +/- 0.6 and 10.1 +/- 0.8, respectively. Extracellular adenosine concentrations increased from (nM, n = 3) 42 +/- 4 in the CG, to 99 +/- 8 in the HGD group and 86 +/- 3 in the RG. Real-time PCR and immunoblotting revealed that in the HGD group and RG, the amounts of rENT1 mRNA and protein were reduced to 40 and 50%, when compared to the CG, respectively. Astrocyte cultures took up [(3)H]adenosine by concentrative and equilibrative transport processes; however, rENT1-mediated uptake was absent in the RG and cultures from the RG took up significantly less [(3)H]adenosine by equilibrative mechanisms than cultures from the CG.

Expression and functional activity of nucleoside transporters in human choroid plexus.

BACKGROUND: Human equilibrative nucleoside transporters (hENTs) 1-3 and human concentrative nucleoside transporters (hCNTs) 1-3 in the human choroid plexus (hCP) play a role in the homeostasis of adenosine and other naturally occurring nucleosides in the brain; in addition, hENT1, hENT2 and hCNT3 mediate membrane transport of nucleoside reverse transcriptase inhibitors that could be used to treat HIV infection, 3'-azido-3'-deoxythymidine, 2'3'-dideoxycytidine and 2'3'-dideoxyinosine. This study aimed to explore the expression levels and functional activities of hENTs 1-3 and hCNTs 1-3 in human choroid plexus. METHODS: Freshly-isolated pieces of lateral ventricle hCP, removed for various clinical reasons during neurosurgery, were obtained under Local Ethics Committee approval. Quantification of mRNAs that encoded hENTs and hCNTs was performed by the hydrolysis probes-based reverse transcription real time-polymerase chain reaction (RT-qPCR); for each gene of interest and for 18 S ribosomal RNA, which was an endogenous control, the efficiency of PCR reaction (E) and the quantification cycle (Cq) were calculated. The uptake of [(3)H]inosine by the choroid plexus pieces was investigated to explore the functional activity of hENTs and hCNTs in the hCP. RESULTS: RT-qPCR revealed that the mRNA encoding the intracellularly located transporter hENT3 was the most abundant, with E(-Cq )value being only about 40 fold less that the E(-Cq )value for 18 S ribosomal RNA; mRNAs encoding hENT1, hENT2 and hCNT3 were much less abundant than mRNA for the hENT3, while mRNAs encoding hCNT1 and hCNT2 were of very low abundance and not detectable. Uptake of [(3)H]inosine by the CP samples was linear and consisted of an Na(+)-dependent component, which was probably mediated by hCNT3, and Na(+)-independent component, mediated by hENTs. The latter component was not sensitive to inhibition by S-(4-nitrobenzyl)-6-thioinosine (NBMPR), when used at a concentration of 0.5 muM, a finding that excluded the involvement of hENT1, but it was very substantially inhibited by 10 muM NBMPR, a finding that suggested the involvement of hENT2 in uptake. CONCLUSION: Transcripts for hENT1-3 and hCNT3 were detected in human CP; mRNA for hENT3, an intracellularly located nucleoside transporter, was the most abundant. Human CP took up radiolabelled inosine by both concentrative and equilibrative processes. Concentrative uptake was probably mediated by hCNT3; the equilibrative uptake was mediated only by hENT2. The hENT1 transport activity was absent, which could suggest either that this protein was absent in the CP cells or that it was confined to the basolateral side of the CP epithelium.

Blood-brain barrier efflux transport of pyrimidine nucleosides and nucleobases in the rat.

The brain efflux index (BEI), a measurement of blood-brain barrier (BBB) efflux transport, was estimated at 15 s, 30 s, 1 min, 3 min and 10 min after intracerebral injection of [14C]pyrimidines. An initial steep increase of the BEI values over time was observed for [14]uracil and [14C]thymine, followed by a more moderate increase after 1 min. For the corresponding nucleosides, [14C]uridine and [14C]thymidine, the increase of BEI values over time was less steep and linear between 30 s and 3 min. The apparent BBB efflux clearances for [14C]uridine, [14C]thymidine, [14C]uracil and [14C]thymine were (microl/min/g): 95.2 +/- 12.1, 125.3 +/- 18.4, 290.4 +/- 28 and 358.5 +/- 32.5, respectively, which is at least several folds higher than the predicted BBB influx clearances of uridine, uracil and thymidine. Quick depletion of brain parenchyma from brain microvasculature has revealed that [14C] radioactivity accumulated in brain microvessels after injection of nucleosides [14C]thymidine and [14C]uridine, but that was not observed when nucleobases, [14C]thymine and [14C]uracil, were injected. Reverse transcriptase-PCR revealed that the rat brain and liver (positive control) express dihydropyrimidine dehydrogenase, a key enzyme in pyrimidine nucleobase catabolism. Two bands representing spliced variants have been detected with the relative density of the bands (expressed relative to the density of glyceraldehyde3-phosphate dehydrogenase bands, mean +/- SEM from 3 separate samples) 0.16 +/- 0.06 and 0.04 +/- 0.01 (brain) and 0.49 +/- 0.1 and 0.07 +/- 0.01 (liver). Overall, these results indicate that the net direction of pyrimidine BBB transport is the efflux transport; rapid BBB efflux transport and metabolic breakdown of pyrimidine nucleobases appear to be important for brain homeostasis.

Quantitative Determination of Cellular-and Neurite Motility Speed in Dense Cell Cultures.

Mobility quantification of single cells and cellular processes in dense cultures is a challenge, because single cell tracking is impossible. We developed a software for cell structure segmentation and implemented 2 algorithms to measure motility speed. Complex algorithms were tested to separate cells and cellular components, an important prerequisite for the acquisition of meaningful motility data. Plasma membrane segmentation was performed to measure membrane contraction dynamics and organelle trafficking. The discriminative performance and sensitivity of the algorithms were tested on different cell types and calibrated on computer-simulated cells to obtain absolute values for cellular velocity. Both motility algorithms had advantages in different experimental setups, depending on the complexity of the cellular movement. The correlation algorithm (COPRAMove) performed best under most tested conditions and appeared less sensitive to variable cell densities, brightness and focus changes than the differentiation algorithm (DiffMove). In summary, our software can be used successfully to analyze and quantify cellular and subcellular movements in dense cell cultures.

Viability and Contractility of Rat Brain Pericytes in Conditions That Mimic Stroke; an in vitro Study.

Reopening of the cerebral artery after occlusion often results in "no-reflow" that has been attributed to the death and contraction (rigor mortis) of pericytes. Since this hypothesis still needs to be confirmed, we explored the effects of oxygen glucose deprivation (OGD) on viability and cell death of primary rat pericytes, in the presence or absence of neurovascular unit-derived cytokines. Two morphodynamic parameters, single cell membrane mobility (SCMM) and fractal dimension (Df), were used to analyze the cell contractions and membrane complexity before and after OGD. We found a marginal reduction in cell viability after 2-6 h OGD; 24 h OGD caused a large reduction in viability and a large increase in the number of apoptotic and dead cells. Application of erythropoietin (EPO), or a combination of EPO and endothelial growth factor (VEGFA1-165) during OGD significantly reduced cell viability; application of Angiopoietin 1 (Ang1) during OGD caused a marginal, insignificant increase in cell viability. Simultaneous application of EPO, VEGFA1-165, and Ang1 significantly increased cell viability during 24 h OGD. Twenty minutes and one hour OGD both significantly reduced SCMM compared to pre-OGD values, while no significant difference was seen in SCMM before and after 3 h OGD. There was a significant decrease in membrane complexity (Df) at 20 min during the OGD that disappeared thereafter. In conclusion, OGD transiently affected cell mobility and shape, which was followed by apoptosis in cultured pericytes. Ang1 may have a potentiality for preventing from the OGD-induced apoptosis. Further studies could clarify the relationship between cell contraction and apoptosis during OGD.

Transport of [14C]hypoxanthine by sheep choroid plexus epithelium as a monolayer in primary culture: Na+-dependent and Na+-independent uptake by the apical membrane and rapid intracellular metabolic conversion to nucleotides.

Hypoxanthine is the main product of purine metabolic degradation and previous studies have revealed that it is present in the sheep CSF and plasma in micromolar concentrations. The aim of this study was to elucidate the transport of this molecule across the sheep choroid plexus epithelium (CPE) as a monolayer in primary culture, to explore the mechanism of uptake by the apical side of the CPE and investigate the metabolic changes inside the cell. The estimated permeability of the CPE monolayer for [14C]hypoxanthine, [14C]adenine and [14C]guanine was low and comparable to the permeability towards the extracellular space markers. The study of [14C]hypoxanthine uptake by the CPE revealed two components: Na+-dependent and Na+-independent, the latter being partially mediated by the equilibrative nucleoside transporter 2. HPLC with simultaneous detection of radioactivity revealed that the majority of [14C]hypoxanthine inside the CPE is metabolised into [14C]nucleotides and [14C]inosine. The remaining intact [14C]hypoxanthine was transported across the opposite, basolateral side of CPE and appeared in the lower chamber buffer together with [14C]inosine. These findings indicate two possible roles of hypoxanthine uptake from the CSF by the CP epithelium in vivo: to provide material for nucleotide synthesis through the salvage pathways in the CPE, as well as to transfer excess hypoxanthine from CSF to blood.

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.

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.

Differential effects of paracrine factors on the survival of cells of the neurovascular unit during oxygen glucose deprivation.

BACKGROUND: Disruption of the neurovascular unit following cerebral ischemia affects protective function of the blood-brain barrier, thus contributing to vasogenic edema and hemorrhagic transformation. AIMS: This study explored the effects of mediators released from neurovascular unit cells on death of brain endothelial cells, astrocytes, pericytes, and microglia during oxygen glucose deprivation. METHODS: Rat primary cell cultures were exposed either to oxygen glucose deprivation or control conditions. Cell death and released angiogenic factors were assessed from media collected from cultures. For some experiments, astrocyte-conditioned media, pericyte-conditioned media, and microglia-conditioned media, collected from the corresponding cell culture after six-hour oxygen glucose deprivation, were added to the media during oxygen glucose deprivation incubations. RESULTS: Brain endothelial cells were more susceptible to death following oxygen glucose deprivation than other neurovascular unit cells. Neither astrocyte-conditioned media nor vascular endothelial growth factor165 were protective for pericytes or brain endothelial cells during oxygen glucose deprivation. Vascular endothelial growth factor receptor antagonist significantly reduced cell death of brain endothelial cells treated with astrocyte-conditioned media or vascular endothelial growth factor165. Pericyte-conditioned media were protective for brain endothelial cells and microglia, but this was not mediated by pericyte-released angiopoietin 1. Soluble angiopoietin 1/angiopoietin 2 receptor Tie2 was protective for brain endothelial cells. Microglia-conditioned media were protective for astrocytes and brain endothelial cells, possibly through transforming growth factor ß1 or interleukin 6. CONCLUSION: Microglia-derived signaling molecules, but not angiogenic factors, were protective for neurovascular unit cells during oxygen glucose deprivation. This finding could identify a potential therapeutic target for ischemic stroke.

The structure of the choroid plexus and the physiology of the choroid plexus epithelium.

The choroid plexuses (CPs) are leaf-like highly vascular structures laying in the ventricles. The main function of choroid plexuses is the production of the cerebrospinal fluid (CSF). Although CPs have a unique distribution of ion transporters/channels, the mechanism of CSF production is similar to the production of fluids in other epithelia and is based on energy released from ATP hydrolysis, which drives unidirectional flux of ions accompanied by movement of water by osmosis. The CPs have an important role in the homeostasis of nutrients in the CSF since the kinetic parameters of glucose and amino acid (AA) transport across the CPs are the main reason for the low concentration of these molecules in the CSF. The CPs appear to be source of CSF-borne hormones and growth factors, including insulin-like growth factor II (IGF II), vasopressin (VP) and transforming growth factor beta1 (TGF-beta1). The CPs also synthesise the thyroid transporting protein transthyretin and transferrin and can chelate heavy metals.

The kinetics of hypoxanthine transport across the perfused choroid plexus of the sheep.

The uptake of principal salvageable nucleobase hypoxanthine was investigated across the basolateral membrane of the sheep choroid plexus (CP) perfused in situ. The results suggest that hypoxanthine uptake was Na+-independent, which means that transport system on the basolateral membrane can mediate the transport in both directions. Although the unlabelled nucleosides adenosine and inosine markedly reduce the transport it seems that this inhibition was due to nucleoside degradation into nucleobases in the cells, since non-metabolised nucleoside analogue NBTI did not inhibit the transport. The presence of adenine also inhibits hypoxanthine uptake while the addition of the pyrimidines does not show any effect, so it seems that the transport of purine nucleobases through basolateral membrane is mediated via a common transporter which is different from the nucleoside transporters. The inclusion of allopurinol in the perfusion fluid did not change the value and general shape of the curve for the uptake which suggest that degradation of hypoxanthine into xanthine and uric acid does not occur in the CP. The capacity of the CP basolateral membrane to transport hypoxanthine is high (90.63+/-3.79 nM/min/g) and close to the values obtained for some essential amino acids by the CP and blood-brain barrier, while the free diffusion is negligible. The derived value of Km (20.72+/-2.42 microM) is higher than the concentration of hypoxanthine in the sheep plasma (15.61+/-2.28 microM) but less than a half of the concentration in the CSF, which indicates that the transport system at basolateral membrane mostly mediates the efflux of hypoxanthine from the cerebrospinal fluid in vivo.

The efflux of purine nucleobases and nucleosides from the rat brain.

The efflux of purine nucleobases and their nucleosides from the rat brain was investigated using the brain efflux index (BEI) method. Calculated BEI values showed that purine nucleobases had very rapid initial efflux after the intracerebral injection, which was followed by the slower efflux due to the intracellular trapping of labelled molecules and confirmed by the capillary depletion technique. The efflux of ribonucleosides was much slower than the efflux of nucleobases and the structure of the sugar moiety seemed to be important, since a significant difference in the efflux velocity between ribo- and deoxyribonucleosides was observed. The results of self- and cross-inhibition studies suggested that the efflux of test molecules was saturable and that purines shared the same transport system on the abluminal side of the blood-brain barrier.

The linkage of glucose to tiazofurin decreases in vitro uptake into rat glioma C6 cells.

The aim of this study was to analyse the uptake of the synthetic nucleoside tiazofurin and glucoso-linker-tiazofurin conjugate (GLTC) into rat C6 glioma cells in vitro. Results indicated that C6 cells accumulated [3H] tiazofurin slowly with time and that accumulation was reduced by the presence of unlabelled GLTC in the medium which implies that GLTC competes with tiazofurin for transport sites. Uptake of [14C] 2 deoxy-glucose into these cells was very rapid and was not affected by the presence of unlabelled GLTC. To prove the true rate of uptake, the HPLC analysis of cellular extract was performed. After the 360 min of incubation in medium that contained 0.15 mM of tiazofurin, the sum of the concentration of tiazofurin and it's metabolite thiazole-adenine dinucleotide (TAD) in the cells was a total of approximately 4.8% of the amount added to each flask. After the same period of incubation in medium which contained 0.15 mM of GLTC, the sum of concentrations of conjugate, free tiazofurin and TAD represented less than 1/3 of the total concentration measured after the incubation with free tiazofurin and was further reduced in the presence of dipyridamole. Therefore, it can be concluded that GLTC shows some affinity for the nucleoside transporter, but the actual rate of uptake is low.

Studies on the human choroid plexus in vitro.

The role of human choroid plexus (CP) epithelium in the transport of solutes between the blood and the cerebrospinal fluid and/or in secretion processes may be studied by employing several experimental approaches. There are a number of in vitro techniques for human CP epithelium (CPE) and all have limitations that do not exclude them a priori, but that should be carefully taken into consideration. Developmental and morphological studies have been largely performed on human choroid plexus samples of either embryonic or post-mortem origin. Functional uptake studies may be performed on pathologically unaltered CP samples obtained during surgical removal of choroid plexus tumors. This approach can be used to explore transport processes mainly across the apical side of the CPE, but cannot be used to study vectorial transport across the CPE. Also, these samples have limited viability. A monolayer of CPE in culture, grown on permeable supports, provides the best available tool to study transport processes or polarized secretion by the CP, but thus far only limited attempts to culture these cells have been published and they mainly include data from neoplastic CPE. A study that used a human papilloma-derived cell line in culture showed that it forms a monolayer with barrier properties, although the cells express pleomorphic and neoplastic features and lack contact inhibition. Other cell cultures express some CPE markers but do not develop tight junctions/barrier properties. This article reviews the main characteristics and limitations of available in vitro methods to study human CPE, which could help researchers choose an appropriate experimental approach for a particular study.

Hydrogen peroxide-induced reduction of delayed rectifier potassium current in hippocampal neurons involves oxidation of sulfhydryl groups.

This study examined the effect of H2O2 on the delayed rectifier potassium current (IKDR) in isolated hippocampal neurons. Whole-cell voltage-clamp experiments were performed on freshly dissociated hippocampal CA1 neurons of SD rats before and after treatment with H2O2. To reveal the mechanism behind H2O2-induced changes in IKDR, cells were treated with different oxidizing and reducing agents. External application of membrane permeable H2O2 reduced the amplitude and voltage-dependence of IKDR in a concentration dependent manner. Desferoxamine (DFO), an iron-chelator that prevents hydroxyl radical (OH) generation, prevented H2O2-induced reduction in IKDR. Application of the sulfhydryl-oxidizing agent 5,5 dithio-bis-nitrobenzoic acid (DTNB) mimicked the effect of H2O2. Sulfhydryl-reducing agents dithiothreitol (DTT) and glutathione (GSH) alone did not affect IKDR; however, DTT and GSH reversed and prevented the H2O2-induced inhibition of IKDR, respectively. Membrane impermeable agents GSH and DTNB showed effects only when added intracellularly identifying intracellular sulfhydryl groups as potential targets for hydroxyl-mediated oxidation. However, the inhibitory effects of DTNB and H2O2 at the positive test potentials were completely and partially abolished by DTT, respectively, suggesting an additional mechanism of action for H2O2, that is not shared by DTNB. In summary, this study provides evidence for the redox modulation of IKDR, identifies hydroxyl radical as an intermediate oxidant responsible for the H2O2-induced decrease in current amplitude and identifies intracellular sulfhydryl groups as an oxidative target.

Effects of omeprazole treatment on nucleoside transporter expression and adenosine uptake in rat gastric mucosa.

Increased adenosine concentration inhibits gastric acid secretion in rat via adenosine A1 and A2A receptors, whereas achlorhydria suppresses A1 and A2A receptor gene expression. This study aimed to examine the effects of omeprazole-induced achlorhydria on the expression and functional activity of nucleoside transporters in rat gastric mucosa. Wistar rats were treated for either 1 or 3 days with 0.4 mmol/kg omeprazole via gavage; controls were treated with vehicle. The expression of nucleoside transporters at the transcript level was explored by quantitative real-time polymerase chain reaction assays; the functional activity of nucleoside transporters in gastric mucosa was explored by observing [3H]adenosine uptake in vitro. Gastric mucosa expressed rat equilibrative nucleoside transporter (rENT) 1 and 2, and rat concentrative nucleoside transporter (rCNT) 1, 2, and 3 at the transcript level, and the estimated values for the threshold cycles for target amplification (Ct) were 31.5 +/- 2, 28.5 +/- 2.1, 32.9 +/- 2.2, 29.1 +/- 2, and 28.9 +/- 2.5, respectively (n = 3 or 4). The Ct value for rat beta-actin was 21.9 +/- 1.8 (n = 4). In vitro uptake of [3H]adenosine by gastric mucosa samples consisted of Na+-dependent and Na+-independent components. One-day omeprazole treatment caused no change in nucleoside transporter mRNA levels or in [3H]adenosine uptake. Three-day omeprazole treatments, however, led to a 12-fold and 17-fold increase in rENT2 and rCNT1 mRNA levels, respectively. Samples taken after 3 days of treatment also took up significantly more [3H]adenosine than did samples from the corresponding control. In conclusion, the possible modification of nucleoside transport activities by changes in intraluminal acidity may have significance as part of a purinergic regulatory feedback mechanism in the control of gastric acid secretion.

The choroid plexus-cerebrospinal fluid system: from development to aging.

The function of the cerebrospinal fluid (CSF) and the tissue that secretes it, the choroid plexus (CP), has traditionally been thought of as both providing physical protection to the brain through buoyancy and facilitating the removal of brain metabolites through the bulk drainage of CSF. More recent studies suggest, however, that the CP-CSF system plays a much more active role in the development, homeostasis, and repair of the central nervous system (CNS). The highly specialized choroidal tissue synthesizes trophic and angiogenic factors, chemorepellents, and carrier proteins, and is strategically positioned within the ventricular cavities to supply the CNS with these biologically active substances. Through polarized transport systems and receptor-mediated transcytosis across the choroidal epithelium, the CP, a part of the blood-CSF barrier (BCSFB), controls the entry of nutrients, such as amino acids and nucleosides, and peptide hormones, such as leptin and prolactin, from the periphery into the brain. The CP also plays an important role in the clearance of toxins and drugs. During CNS development, CP-derived growth factors, such as members of the transforming growth factor-beta superfamily and retinoic acid, play an important role in controlling the patterning of neuronal differentiation in various brain regions. In the adult CNS, the CP appears to be critically involved in neuronal repair processes and the restoration of the brain microenvironment after traumatic and ischemic brain injury. Furthermore, recent studies suggest that the CP acts as a nursery for neuronal and astrocytic progenitor cells. The advancement of our knowledge of the neuroprotective capabilities of the CP may therefore facilitate the development of novel therapies for ischemic stroke and traumatic brain injury. In the later stages of life, the CP-CSF axis shows a decline in all aspects of its function, including CSF secretion and protein synthesis, which may in themselves increase the risk for development of late-life diseases, such as normal pressure hydrocephalus and Alzheimer's disease. The understanding of the mechanisms that underlie the dysfunction of the CP-CSF system in the elderly may help discover the treatments needed to reverse the negative effects of aging that lead to global CNS failure.

Polarized distribution of nucleoside transporters in rat brain endothelial and choroid plexus epithelial cells.

This study investigated mRNA expression and protein localization of equilibrative and concentrative nucleoside transporters (ENTs, CNTs) in primary cultures of rat brain endothelial cells (RBEC) and rat choroid plexus epithelial cells (RCPEC). Reverse transcriptase PCR analysis revealed that RBEC and RCPEC contained mRNA for rENT1, rENT2 and rCNT2 and for rENT1, rENT2, rCNT2 and rCNT3, respectively. Immunoblotting of membrane fractions of RBEC, fresh RCPEC and primary cultures of RCPEC revealed the presence of rENT1, rENT2 and rCNT2 proteins in all samples. Measurement of [14C]adenosine uptake into cells grown as monolayers on permeable plastic supports revealed a polarized distribution of Na+-dependent adenosine uptake in that CNT activity was associated exclusively in membranes of RBEC facing the lower chamber (which corresponds to the surface facing the interstitial fluid in situ) and in membranes of RCPEC facing the upper chamber (which corresponds to the surface facing the cerebrospinal fluid in situ). In both RBEC and RCPEC, adenosine uptake from the opposite chambers was Na+-independent and partially inhibited by nitrobenzylthioinosine, indicating the presence of the equilibrative sensitive transporter rENT1.

Brain to blood efflux transport of adenosine: blood-brain barrier studies in the rat.

The blood-brain barrier (BBB) efflux transport of [(14)C] adenosine was studied using the brain efflux index (BEI) technique. BEI increased linearly over the first 2 min after injection, with deviation from linearity thereafter; 90.12 +/- 1.5% of the injected [(14)C] radioactivity remained within the brain after 20 min. The remaining tracer appears to be mainly intracellular, trapped by phosphorylation, as an almost linear increase of BEI over 20 min was observed after intracerebral injection of [(14)C] adenosine together with 5-iodo tubercidin. The BBB efflux clearance of [(14)C] radioactivity was estimated to be 27.62 +/- 5.2 micro L/min/g, almost threefold higher than the BBB influx clearance estimated by the brain uptake index technique. High-performance liquid chromatography (HPLC) analysis of blood plasma collected from the jugular vein after the intracerebral injection revealed metabolic breakdown of [(14)C] adenosine into nucleobases. The BBB efflux transport was saturable with apparent K(m) = 13.22 +/- 1.75 micro m and V(max) = 621.07 +/- 71.22 pmole/min/g, which indicated that BBB efflux in vivo is 6.2-12p mole/min/g, negligible when compared to the reported rate of adenosine uptake into neurones/glia. However, these kinetic parameters also suggest that under conditions of elevated ISF adenosine in hypoxia/ischaemia, BBB efflux transport could increase up to 25% of the uptake into neurones/glia and become an important mechanism to oppose the rise in ISF concentration. HPLC-fluorometry detected 93.6 +/- 5.25 nm of adenosine in rat plasma, which is 17- to 220-fold lower than the reported K(m) of adenosine BBB influx in rat. Together with the observed rapid degradation inside endothelial cells, this indicated negligible BBB influx of intact adenosine under resting conditions. Cross-inhibition studies showed that unlabelled inosine, adenine and hypoxanthine caused a decrease in BBB efflux of [(14)C] radioactivity in a concentration-dependent manner, with K(i) of 16.7 +/- 4.88, 65.1 +/- 14.1 and 71.1 +/- 16.9 micro m, respectively. This could be due to either competition of unlabelled molecules with [(14)C] adenosine or competition with its metabolites hypoxanthine and adenine for the same transport sites.