Zoran B. Redzic

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

The blood–brain barrier (BBB) efflux transport of [14C] 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 [14C] 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 [14C] adenosine together with 5‐iodo tubercidin. The BBB efflux clearance of [14C] radioactivity was estimated to be 27.62 ± 5.2 µL/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 [14C] adenosine into nucleobases. The BBB efflux transport was saturable with apparent Km = 13.22 ± 1.75 µm and Vmax = 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 Km 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 [14C] radioactivity in a concentration‐dependent manner, with Ki of 16.7 ± 4.88, 65.1 ± 14.1 and 71.1 ± 16.9 µm, respectively. This could be due to either competition of unlabelled molecules with [14C] adenosine or competition with its metabolites hypoxanthine and adenine for the same transport sites.

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 characteristics of nucleobase transport and metabolism by the perfused sheep choroid plexus

The uptake of nucleobases was investigated across the basolateral membrane of the sheep choroid plexus perfused in situ. The maximal uptake (U(max)) for hypoxanthine and adenine, was 35.51+/-1.50% and 30.71+/-0.49% and for guanine, thymine and uracil was 12.00+/-0.53%, 13.07+/-0.48% and 12.30+/-0.55%, respectively with a negligible backflux, except for that of thymine (35.11+/-5.37% of the U(max)). HPLC analysis revealed that the purine nucleobase hypoxanthine and the pyrimidine nucleobase thymine can pass intact through the choroid plexus and enter the cerebrospinal fluid CSF so the lack of backflux for hypoxanthine was not a result of metabolic trapping in the cell. Competition studies revealed that hypoxanthine, adenine and thymine shared the same transport system, while guanine and uracil were transported by a separate mechanism and that nucleosides can partially share the same transporter. HPLC analysis of sheep CSF collected in vivo revealed only two nucleobases were present adenine and hypoxanthine; with an R(CSF/Plasma) 0.19+/-0.02 and 3.43+/-0.20, respectively. Xanthine and urate, the final products of purine catabolism, could not be detected in the CSF even in trace amounts. These results suggest that the activity of xanthine oxidase in the brain of the sheep is very low so the metabolic degradation of purines is carried out only as far as hypoxanthine which then accumulates in the CSF. In conclusion, the presence of saturable transport systems for nucleobases at the basolateral membrane of the choroidal epithelium was demonstrated, which could be important for the distribution of the salvageable nucleobases, adenine and hypoxanthine in the central nervous system.

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

BackgroundEfflux 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.MethodsTo 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 conclusionCPEC 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 Ω 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.

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 kinetics of hypoxanthine efflux from the rat brain

The brain efflux of radiolabelled hypoxanthine in the rat was rapid in the first minute after injection [K(eff)(i)=0.21+/-0.06 min(-1)], which was saturable with a V(max)=13.08+/-0.81 nM min(-1) g(-1), and a high K(m,app) (67.2+/-13.4 microM); the K(i,app) for inosine was 31.5+/-7.6 microM. Capillary depletion analysis indicated that hypoxanthine accumulates in neurons and glia with the time. From cross-inhibition studies with different purines and pyrimidines, it suggests that these molecules could also be important substrates for this carrier.

The effects of NO synthesis inhibition on the uptake of endogenous nucleosides into the rat brain

Brain uptake of 3H endogenous nucleosides in rats was measured by Brain Uptake Index method, using 14C-sucrose as a referent molecule. The values obtained in control group were 4.68±0.93 for adenosine, 4.10±0.72 for guanosine and 1.14±0.72 for thymidine, indicating significant blood-to-brain transport of purine nucleosides. NO-synthase inhibition was induced by i.p. application of 25 mg/kg 1-ω-nitro-arginin methyl ester (L-NAME) and 5mg/kg diphenylethiodonium (brain parenchyma NOS inhibitor). HPLC analysis showed that the concentration of L-NAME in brain was sufficient to cause inhibition of NOS. Application of L-NAME resulted in significant inhibition of brain uptake for both purine nucleosides. Unlike adenosine, in the case of guanosine a correlation between the rate of brain uptake inhibition and the L-NAME concentration in plasma, but not in brain, was obtained. Application of diphenylethiodonium also caused a significant decrease in both guanosine and adenosine brain uptake. The obtained results were not significantly different from the BUI values obtained after preapllication of L-NAME. Our results suggest that NOS inhibition decreases brain uptake of purine nucleosides. Such effect(s) are probably due to the inhibition of brain parenchyma NOS.

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 its 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.

Transport Mechanisms of Nucleosides and Nucleoside Analogues ReverseTranscriptase Inhibitors in the Brain

Because of their hydrophilic natures, movements of nucleosides and many of their analogues across cellular membranes are mediated by nucleoside transporter (NT) proteins. Thus, NT proteins play an important role in physiological actions of nucleosides and in alterations of their function under various pathophysiological conditions. Also, these proteins are important for the therapeutic actions of some of synthetic nucleosides that have pharmacological actions and are thus used as drugs, including nucleoside analogues reverse transcriptase inhibitors (King et al., 2006, Zhang et al., 2007). Before the “molecular biology” era, it was possible to explore nucleoside transport processes mainly by functional transport studies, which measured transcellular flux or cellular uptake of radiolabelled nucleosides. These early studies have identified two distinct transport processes in mammalian cells: the equilibrative bidirectional transport with lower affinity for naturally occurring nucleosides and the concentrative, unidirectional secondary active transport (Na+/nucleoside cotransport) which revealed higher affinity for nucleosides (Hyde et al., 2004, Baldwin et al., 2004). Also, based on inhibition of these processes by synthetic analogues and based on substrate specificity and kinetics, it was recognized that both groups were heterogeneous: equilibrative nucleoside transport processes were further categorized as either nitrobenzylthioinosine (NBMPR)-sensitive (es) or NBMPR-insensitive (ei), while concentrative transport processes were categorized as either cit (concentrative, NBMPR insensitive, thymidine important substrate), cif (concentrative, NBMPR insensitive, formycin B important substrate) or cib (concentrative, NBMPR insensitive, broadly selective for purine and pyrimidine nucleosides) (Cass et al., 1998). Transport studies have also revealed that all these transport processes are not ubiquitously distributed in all mammalian cells; while equlibrative transport processes were more or less ubiquitous, concentrative transport processes were mainly found in epithelia, endothelial layers and in the liver (for an early review of nucleoside transport processes see Young and Jarvis 1983). Development of molecular biology techniques has allowed identification of the proteins responsible for nucleoside transport processes (King et al., 2006). Purification and N-terminal sequencing of the es transporter from human erythrocytes enabled cloning a human placental

Transport of 3H L-Alanine Across the Blood-Brain Barrier of in Situ Perfused Guinea Pig Brain

Transport of 3H L-alanine through the blood-brain barrier (BBB) was studied using brain vascular perfusion method in guinea pig. Our results indicate that L-alanine passes across the luminal side of the BBB. Unidirectional transport constant Kin ranged from 4.871±0.622 μmin−1g−1 in hippocampus to 5.608±0.902 μmin−1g−1 in parietal cortex, which is comparable with the values obtained for other small neutral non-essential amino acids. Addition of unlabelled L-alanine to perfusing medium caused the decrease in L-alanine transport, indicating the importance of saturable component for L-alanine transport. However, presence of high concentrations of unlabelled L-alanine in perfusing medium (up to 12 mmol/l), did not result in complete inhibition of 3H L-alanine transport through the BBB. Therefore, it seems that another mechanism is also involved in 3H L-alanine transport across the endothelial cells’ luminal membrane. Values for Michaelis-Menten constant for L-alanine transport from blood into brain point out that the affinity of this molecule to its carrier(s) is rather small (Km >1 mmol/1). Capacity of 3H L-alanine blood-to-brain transport is very small as well (Vmax <20 nmol/min/g).