Nicotine and cotinine increases the brain penetration of saquinavir in rat

Endothelial tight junctions and efflux transporters of the blood-brain barrier (BBB) significantly limit brain accumulation of many drugs, including protease inhibitors such as saquinavir. The cholinergic agonist nicotine is one of the most commonly used drugs in the world and the incidence is even higher in the human immune deficiency virus population (～ 70%). We examined the ability of nicotine and its primary metabolite cotinine to modify brain uptake of saquinavir in rats. Both nicotine and cotinine at pharmacological concentrations matching those in smokers, increased brain saquinavir uptake by two fold. Co-perfusion with nicotinic receptor antagonists and passive permeability markers showed that the effect was not caused by receptor activation or BBB permeability disruption. Transport inhibition studies demonstrated that brain saquinavir uptake is limited by multiple efflux transporters, P-glycoprotein (P-gp), breast cancer resistance protein and multidrug resistance-associated protein. In situ perfusion and in vitro experiments using a classical P-gp substrate rhodamine 123 linked the effect of nicotine to inhibition of BBB P-gp transport. The effect was confirmed in vivo in chronic 14 day nicotine administration animals. These data suggest nicotine increases antiretroviral drug exposure to brain and may represent a significant in vivo drug-drug interaction at the BBB. Although this may slightly benefit CNS antiretroviral efficacy, it may also expose the brain to potential serious neurotoxicity.     

A bioreversible prodrug approach designed to shift mechanism of brain uptake for amino-acid-containing anticancer agents

By derivatization at the N-terminus of amino acid-based anticancer agents (e.g. melphalan and acivicin) to form a drug delivery system (TDDS), we demonstrate a change in the mechanism of brain uptake from the large neutral amino acid transporter (LAT) pathway to passive. An in situ rat brain perfusion technique was used to determine the brain capillary permeability-surface area (PA) product for [(14)C]L-Leu as control (5.18 +/- 0.32 x 10(-2) mL/s/g), which was inhibited competitively (to 7-18% of control) by an excess concentration of the amino-acid-containing anticancer agents, acivicin and melphalan. However, TDDS did not compete for LAT-mediated brain uptake of the radiotracer [(14)C]L-Leu. Brain uptake of TDDS was determined after in situ brain perfusion followed by RP-HPLC along with LC-MS/MS detection of the analytes in brain samples. The PA product for CH(3)-TDDS containing melphalan (5.09 +/- 2.0 x 10(-2) mL/s/g) shows that these agents rapidly cross the blood-brain barrier. Furthermore, competition studies of CH(3)-TDDS with [(3)H]verapamil suggest that the TDDS interacts significantly with the multidrug resistant efflux system (P-glycoprotein) at the blood-brain barrier. Therefore, TDDS were shown to lack LAT-mediated brain uptake. The drug delivery systems, however, showed uptake predominantly via the passive route along with recognition by the multidrug resistant efflux protein at the cerebrovasculature.     

Nicotine Exposure Does not Alter Plasma to Brain Choline Transfer

Acute and chronic nicotine exposure in rats is associated with an increase in brain acetylcholine (ACh) transmission. The acquisition of choline for neuronal ACh synthesis occurs primarily via two pathways; first, free choline is transported from the blood across the blood-brain barrier (BBB) and/or second, from synaptic choline generated by either hydrolysis of non-bound ACh or membrane phosphatidylcholine catabolism. To determine if nicotine-induced cholinergic demand is associated with increased choline transport rates into brain, we measured BBB choline transport in naïve and S-(−) nicotine exposed rats (acute and chronic, 4.5 mg/kg/d for 1, 14, 21 and 28 d; osmotic minipumps) using the in situ rat brain perfusion technique. No significant changes in choline uptake after acute or chronic nicotine exposure were observed in whole brain or cortex. Of considerable interest was a significant decrease in regional brain choline uptake measured in the hippocampus after chronic nicotine exposure (28 d). Our data suggest that the increased ACh transmission observed after nicotine exposure does not correlate with increased blood-to-brain transfer of choline. Considering these data and previous literature reports, we propose that the additional free choline required under conditions of nicotine exposure (for ACh synthesis) is primarily recruited from membrane phospholipid metabolism.     

Characterization of the blood-brain barrier choline transporter using the in situ rat brain perfusion technique

Choline enters brain by saturable transport at the blood-brain barrier (BBB). In separate studies, both sodium-dependent and passive choline transport systems of differing affinity have been reported at brain capillary endothelial cells. In the present study, we re-examined brain choline uptake using the in situ rat brain perfusion technique. Saturable brain choline uptake from perfusion fluid was best described by a model with a single transporter (V:(max) = 2.4-3.1 nmol/min/g; K(m) = 39-42 microM) with an apparent affinity (1/Km)) for choline five to ten-fold greater than previously reported in vivo, but less than neuronal 'high-affinity' brain choline transport (K(m) = 1-5 microM). BBB choline uptake from a sodium-free perfusion fluid using sucrose for osmotic balance was 50% greater than in the presence of sodium suggesting that sodium is not required for transport. Hemicholinium-3 inhibited brain choline uptake with a K(i) (57 +/- 11 microM) greater than that at the neuronal choline system. In summary, BBB choline transport occurs with greater affinity than previously reported, but does not match the properties of the neuronal choline transporter. The V:(max) of this system is appreciable and may provide a mechanism for delivering cationic drugs to brain.     

Interaction between nicotine and MPTP/MPP+ in rat brain endothelial cells

To examine the interaction between nicotine and MPTP/MPP+ in the blood-brain barrier, cellular uptake of MPTP and MPP+ was studied in the presence of nicotine and several compounds, including MPTP/MPP+ analogs and a specific inhibitor of organic cation transporter (OCT) in an adult rat brain microvascular endothelial cell line (ARBEC). The kinetic properties of the uptake of MPTP, MPP+, and nicotine were also examined. In addition, a microdialysis study was performed to evaluate the in vivo effect of nicotine (i.p.) on extracellular levels of MPTP and MPP+ in the brain after intravenous administration of MPTP. The results showed that uptake of MPTP, MPP+, and nicotine was partly mediated by a carrier system that was sensitive to decynium22, a specific OCT inhibitor. RT-PCR showed the presence of OCT1 mRNA in ARBEC. Capacity for uptake of MPTP and nicotine was much higher than that for MPP+ (Km and Vm values of 10.94+/-1.44 microM and 0.049+/-0.007 pmol/mg s, respectively, for MPP+, compared to values of 35.75+/-0.85 microM and 40.95+/-3.56 pmol/mg s for MPTP and 25.29+/-6.44 microM and 51.15+/-14.18 pmol/mg s for nicotine). In addition, nicotine competitively inhibited the uptake of both MPTP and MPP+, with inhibition constants (Ki) of 328 microM and 210 microM, respectively. In vivo microdialysis results showed that nicotine significantly reduced brain extracellular levels of MPTP in the first 30 min (507.4+/-8.5 ng/ml vs. 637.9+/-30.8 ng/ml with and without nicotine pre-treatment, respectively), but did not have significant effect on those of MPP+. In conclusion, nicotine can inhibit in vitro cellular uptake and in vivo transfer of MPTP across the blood-brain barrier, which can be mediated by multiple pathways including OCT1.     

Inhibition of the rat blood–brain barrier choline transporter by manganese chloride

Choline transport has been characterized by multiple mechanisms including the blood-brain barrier (BBB), and high- and low-affinity systems. Each mechanism has unique locations and characteristics yet retain some similarities. Previous studies have demonstrated cationic competition by monovalent cations at the BBB and cation divalent manganese in the high-affinity system. To evaluate the effects of divalent manganese inhibition as well as other cationic metals at the BBB choline transporter, brain choline uptake was evaluated in the presence of certain metals of interest in Fischer-344 rats using the in situ brain perfusion technique. Brain choline uptake was inhibited in the presence of Cd(2+) (73 +/- 2%) and Mn(2+) (44 +/- 6%), whereas no inhibition was observed with Cu(2+) and Al(3+). Furthermore, it was found that manganese caused a reduction in brain choline uptake and significant regional choline uptake inhibition in the frontal and parietal cortex, the hippocampus and the caudate putamen (45 +/- 3%, 68 +/- 18%, 58 +/- 9% and 46 +/- 15%, respectively). These results suggest that choline uptake into the CNS can be inhibited by divalent cationic metals and monovalent cations. In addition, the choline transporter may be a means by which manganese enters the brain.     

Functional expression of the serotonin transporter in immortalized rat brain microvessel endothelial cells

There is evidence from recent studies that the brain endothelium (of capillaries and/or larger vessels) may serve as a specific target for serotonin [5-hydroxytryptamine (5-HT)]. This neurotransmitter is expected to be involved in the regulation of the blood-brain barrier (BBB) permeability and/or of the cerebral blood flow via receptor-mediated mechanisms. Effective control of these processes depends on a speedy uptake and metabolism of released 5-HT molecules. To realize this, a similar mechanism of 5-HT uptake as in brain may exist at the BBB. In this study, we have demonstrated using RT-PCR that 5-HT transporter mRNA is present in the brain endothelium and that a saturable transport system for 5-HT is functionally expressed in immortalized rat brain endothelial cells (RBE4 cells). These cells take up [3H]5-HT by an active saturable process with a Km value of 397 +/- 64 nmol/L and a transport capacity of 51.7 +/- 3.5 pmol x g(-1) x min(-1). The 5-HT uptake depends on Na+, as indicated by the replacement of NaCl by LiCl. The 5-HT uptake was sensitive to specific 5-HT transport inhibitors such as paroxetine, clomipramine, fluoxetine, and citalopram but not to inhibitors of the vesicular amine transporter such as reserpine or tetrabenazine. Our results demonstrate that cerebral endothelial cells are able to participate actively in the removal and metabolism of the released 5-HT, which supports the concept of direct serotoninergic regulation of the BBB function.     

Efflux of a suppressive neurotransmitter, GABA, across the blood-brain barrier

In this study, GABA efflux transport from brain to blood was estimated by using the brain efflux index (BEI) method. [3H]GABA microinjected into parietal cortex area 2 (Par2) of the rat brain was eliminated from the brain with an apparent elimination half-life of 16.9 min. The blood-brain barrier (BBB) efflux clearance of [3H]GABA was at least 0.153 mL/min/g brain, which was calculated from the elimination rate constant (7.14 x 10(-2) x min(-1)) and the distribution volume in the brain (2.14 mL/g brain). Direct comparison of the apparent BBB influx clearance [3H]GABA (9.29 microL/min/g brain) and the apparent efflux clearance (153 microL/min/g brain) indicated that the efflux clearance was at least 16-fold greater than the influx clearance. In order to reduce the effect of metabolism in the neuronal cells following intracerebral microinjection, we determined the apparent efflux of [3H]GABA in the presence of nipecotic acid, a GABA transport inhibitor in parenchymal cells, using the BEI method. Under such conditions, the elimination of [3H]GABA across the BBB showed saturation and inhibition by probenecid in the presence of nipecotic acid. Furthermore, the uptake of [3H]GABA by MBEC4 cells was inhibited by GABA, taurine, beta-alanine and nipecotic acid in a concentration-dependent manner. It is likely that GABA inhibits the first step in the abluminal membrane uptake by brain endothelial cells, and that probenecid selectively inhibits the luminal membrane efflux transport process from the brain capillary endothelial cells based on the in vivo and in vitro evidence. The BBB acts as the efflux pump for GABA to reduce the brain interstitial fluid concentration.     

Brain-to-blood efflux transport of estrone-3-sulfate at the blood-brain barrier in rats

Efflux transport of estrogens such as estrone-3-sulfate (E1S), and estrone (E1) across the blood-brain barrier (BBB) was evaluated using the Brain Efflux Index (BEI) method. The apparent BBB efflux rate constant (Keff) of [3H]E1S, and [3H]E1 was 6.63 x 10(-2) +/- 0.77 x 10(-2) min(-1), and 6.91 x 10(-2) +/- 1.23 x 10(-2) min(-1), respectively. The efflux transport of [3H]E1S from brain across the BBB was a saturable process with Michaelis constant (Km) of 96.0 +/- 34.4 microM and 93.4 +/- 22.0 microM estimated by two different methods. By determining [3H]E1S metabolites using high performance liquid chromatography (HPLC) after intracerebral injection, significant amounts of [3H]E1S were found in the jugular venous plasma, providing direct evidence that most of [3H]E1S is transported from brain across the BBB in intact form. To compare the apparent efflux clearance across the BBB of E1S with that of E1, the brain distribution volume of E1S and E1 was estimated using the brain slice uptake method. The apparent efflux clearance of [3H]E1S was determined to be 74.9 +/- 3.8 microl/(min x g brain) due to the distribution volume of 1.13 +/- 0.06 ml/g brain. By contrast, the apparent efflux clearance of E1 was more than 227 +/- 3 microl/(min x g brain), since the distribution volume of [3H]E1 at 60 min was 3.28 +/- 0.13 ml/g. The E1S efflux transport process was inhibited by more than 40% by coadministration of bile acids (taurocholate, and cholate), and organic anions (sulfobromophthalein, and probenecid), whereas other organic anions did not affect the E1S efflux transport. The [3H]E1S efflux was significantly reduced by 48.6% after preadministration of 5 mM dehydroepiandrosterone sulfate. These results suggest that E1S is transported from brain to the circulating blood across the BBB via a carrier-mediated efflux transport system.     

Blood-brain barrier tight junctions are altered during a 72-h exposure to lambda-carrageenan-induced inflammatory pain

In this study, we examined the effect of lambda-carrageenan-induced inflammatory pain on the functional and structural properties of the rat blood-brain barrier (BBB) over a 72-h time period. Systemic inflammation was induced by an intraplantar injection of 3% lambda-carrageenan into the right hind paw of female Sprague-Dawley rats. In situ brain perfusion and Western blot analyses were performed at 1, 3, 6, 12, 24, 48, and 72 h. In situ brain perfusion showed lambda-carrageenan significantly increased brain uptake of [(14)C]sucrose at 1, 3, 6, and 48 h (139 +/- 9%, 166 +/- 19%, 138 +/- 13%, and 146 +/- 7% compared with control, respectively). Capillary depletion analysis insured the increased brain uptake was due to increased BBB permeability and not vascular trapping. Western blot analyses for zonula occludens-1 (ZO-1) and occludin were performed on isolated cerebral microvessels. ZO-1 expression was significantly increased at 1, 3, and 6 h and returned to control expression levels by 12 h. Total occludin expression was significantly reduced at 1, 3, 6, 12, and 48 h. This investigation demonstrated that lambda-carrageenan-induced inflammatory pain elicits a biphasic increase in BBB permeability with the first phase occurring from 1-6 h and the second phase occuring at 48 h. Furthermore, changes in BBB function are correlated with altered tight junctional protein expression of occludin and ZO-1. Changes in the structure of tight junctions may have important clinical ramifications concerning central nervous system homeostasis and therapeutic drug delivery.     

Blood–Brain Barrier Transport of Kynurenines: Implications for Brain Synthesis and Metabolism

To evaluate the potential contribution of circulating kynurenines to brain kynurenine pools, the rates of cerebral uptake and mechanisms of blood-brain barrier transport were determined for several kynurenine metabolites of tryptophan, including L-kynurenine (L-KYN), 3-hydroxykynurenine (3-HKYN), 3-hydroxyanthranilic acid (3-HANA), anthranilic acid (ANA), kynurenic acid (KYNA), and quinolinic acid (QUIN), in pentobarbital-anesthetized rats using an in situ brain perfusion technique. L-KYN was found to be taken up into brain at a significant rate [permeability-surface area product (PA) = 2-3 x 10(-3) ml/s/g] by the large neutral amino acid carrier (L-system) of the blood-brain barrier. Best-fit estimates of the Vmax and Km of saturable L-KYN transfer equalled 4.5 x 10(-4) mumol/s/g and 0.16 mumol/ml, respectively. The same carrier may also mediate the brain uptake of 3-HKYN as D,L-3-HKYN competitively inhibited the brain transfer of the large neutral amino acid L-leucine. For the other metabolites, uptake appeared mediated by passive diffusion. This occurred at a significant rate for ANA (PA, 0.7-1.6 x 10(-3) ml/s/g), and at far lower rates (PA, 2-7 x 10(-5) ml/s/g) for 3-HANA, KYNA, and QUIN. Transfer for KYNA, 3-HANA, and ANA also appeared to be limited by plasma protein binding. The results demonstrate the saturable transfer of L-KYN across the blood-brain barrier and suggest that circulating L-KYN, 3-HKYN, and ANA may each contribute significantly to respective cerebral pools. In contrast, QUIN, KYNA, and 3-HANA cross the blood-brain barrier poorly, and therefore are not expected to contribute significantly to brain pools under normal conditions.     

Blood—Brain Barrier Glucose Transporter

Abstract : The transport of glucose across the blood-brain barrier (BBB) is mediated by the high molecular mass (55-kDa) isoform of the GLUT1 glucose transporter protein. In this study we have utilized the tritiated, impermeant photolabel 2-N-[4-(1-azi-2,2,2-trifluoroethyl)[2-³H]propyl]-1,3-bis(d -mannose-4-yloxy)-2-propylamine to develop a technique to specifically measure the concentration of GLUT1 glucose transporters on the luminal surface of the endothelial cells of the BBB. We have combined this methodology with measurements of BBB glucose transport and immunoblot analysis of isolated brain microvessels for labeled luminal GLUT1 and total GLUT1 to reevaluate the effects of chronic hypoglycemia and diabetic hyperglycemia on transendothelial glucose transport in the rat. Hypoglycemia was induced with continuous-release insulin pellets (6 U/day) for a 12- to 14-day duration ; diabetes was induced by streptozotocin (65 mg/kg i.p.) for a 14- to 21-day duration. Hypoglycemia resulted in 25-45% increases in regional BBB permeability-surface area (PA) values for d -[¹⁴C]glucose uptake, when measured at identical glucose concentration using the in situ brain perfusion technique. Similarily, there was a 23 ± 4% increase in total GLUT1/mg of microvessel protein and a 52 ± 13% increase in luminal GLUT1 in hypoglycemic animals, suggesting that both increased GLUT1 synthesis and a redistribution to favor luminal transporters account for the enhanced uptake. A corresponding (twofold) increase in cortical GLUT1 mRNA was observed by in situ hybridization. In contrast, no significant changes were observed in regional brain glucose uptake PA, total microvessel 55-kDa GLUT1, or luminal GLUT1 concentrations in hyperglycemic rats. There was, however, a 30-40% increase in total cortical GLUT1 mRNA expression, with a 96% increase in the microvessels. Neither condition altered the levels of GLUT3 mRNA or protein expression. These results show that hypoglycemia, but not hyperglycemia, alters glucose transport activity at the BBB and that these changes in transport activity result from both an overall increase in total BBB GLUT1 and an increased transporter concentration at the luminal surface.     

Brain-type glucose transporter (GLUT-1) is selectively localized to the blood-brain barrier. Studies with quantitative western blotting and in situ hybridization

The hypothesis that the GLUT-1 glucose transporter isoform is expressed selectively in brain at the capillary endothelium, i.e. the blood-brain barrier (BBB), was tested by using quantitative Western blotting, cytochalasin B binding, and in situ hybridization in bovine brain cortex. Purified human red cell glucose transporter was used as the standard for quantitative Western blots, because the mobility of the human erythrocyte and BBB glucose transporters in electrophoretic gels was identical. The concentration of immunoreactive glucose transporter in bovine BBB plasma membranes was 10.8 +/- 0.9 pmol/mgp (mean +/- S.E., n = 6). This value was not statistically different from the estimate of the maximal binding sites of D-glucose-displaceable [3H]cytochalasin B binding in the BBB membrane preparations, 11.7 +/- 3.5 pmol/mgp. In situ hybridization experiments using 35S-labeled antisense and sense riboprobes corresponding to nucleotides 385-932 of the GLUT-1 cDNA showed prominent hybridization of the antisense probe over brain microvascular endothelium, but no hybridization over neuropil greater than that found with the 35S-labeled sense probe. These studies are consistent with the following conclusion: (a) essentially 100% of the glucose transporter binding sites at the BBB can be accounted for by the GLUT-1 isoform; (b) in situ hybridization studies confirm previous Northern blot analysis and indicate the GLUT-1 gene is expressed selectively in microvascular endothelium in brain with minimal, if any, expression of this gene in neurons or glial cells in vivo.     

Influence of breast cancer resistance protein (Abcg2) and p-glycoprotein (Abcb1a) on the transport of imatinib mesylate (Gleevec) across the mouse blood-brain barrier

Imatinib, a protein tyrosine kinase inhibitor, may prevent the growth of glioblastoma cells. Unfortunately, its brain distribution is restricted by p-glycoprotein (p-gp or multidrug resistance protein Mdr1a), and probably by breast cancer resistance protein (Bcrp1), two efflux pumps expressed at the blood-brain barrier (BBB). We have used in situ brain perfusion to investigate the mechanisms of imatinib transport across the mouse BBB. The brain uptake of imatinib in wild-type mice was limited by saturable efflux processes. The inhibition of p-gp, by valspodar and zosuquidar, increased imatinib uptake (2.5-fold), as did the deficiency of p-gp in Mdr1a/1b(-/-) mice (5.5-fold). Perfusing imatinib with the p-gp/Bcrp1 inhibitor, elacridar, enhanced the brain uptake of imatinib in wild-type (4.1-fold) and Mdr1a/1b(-/-) mice (1.2-fold). However, the brain uptake of imatinib was similar in wild-type and Bcrp1(-/-) mice when it was perfused at a non-saturating concentration. The brain uptake of CGP74588, an active metabolite of imatinib, was low. It was increased by perfusion with elacridar (twofold), but not with valspodar and zosuquidar. CGP74588 uptake was 1.5 times greater in Bcrp1(-/-) mice than in wild-type mice. These data suggest that imatinib transport at the mouse BBB is limited by p-gp and probably by Bcrp1, and that CGP74588 transport is restricted by Bcrp1.     

Urea cycle disorders, hyperammonemia and neurotransmitter changes

In congenital urea cycle disorders, detoxification of ammonia is impaired, leading to hyperammonemia. Ammonia is the major component causing the acute neurological disturbances. It may influence the supply of substrate and its transport at the blood-brain barrier (BBB) which results in alterations in the synthesis and catabolism of neurotransmitters in the brain. In hyperammonemic rats, the uptake of tryptophan into the brain is increased with an augmented flux through the serotonin pathway. In the forebrain, glutamine as well as amino acids transported with the same L-carrier system, such as phenylalanine, tyrosine and tryptophan, are elevated. It is postulated that the increased transport of tryptophan at the BBB occurs in exchange with glutamine. Methionine sulfoximine (MSO) inhibits glutamine synthetase in the cerebral cortex. The activity drops from 5.85 +/- 0.38 to 1.07 +/- 0.37 mumol/min/g wet weight. Under MSO, the brain tryptophan uptake also decreased to 64.2 +/- 4.5% in hyperammonemic rats, to 54.1 +/- 8.0% in untreated hyperammonemic rats, whereas without MSO an increase of tryptophan uptake was observed. An effect of glutamine on tryptophan transport could also be demonstrated using brain microvessel preparations as a model for the BBB. Our findings indicate that preloading isolated microvessels with L-glutamine increases tryptophan uptake into the endothelia when L-glutamine is at concentrations found in brain homogenates under hyperammonemia. Since brain microvessels do not contain glutamine synthetase activity, enzymes from the gamma-glutamyl cycle may be involved in the glutamine-mediated tryptophan transport.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Transforming Growth Factor-β1 Upregulates the Tight Junction and P-glycoprotein of Brain Microvascular Endothelial Cells

1. The present study was aimed at elucidating effects of transforming growth factor-beta (TGF-beta) on blood-brain barrier (BBB) functions with mouse brain capillary endothelial (MBEC4) cells. 2. The permeability coefficients of sodium fluorescein and Evans blue albumin for MBEC4 cells and the cellular accumulation of rhodamine 123 in MBEC4 cells were dose-dependently decreased after a 12-h exposure to TGF-beta1 (0.01-10 ng/mL). 3. The present study demonstrates that TGF-beta lowers the endothelial permeability and enhances the functional activity of P-gp, suggesting that cellular constituents producing TGF-beta in the brain may keep the BBB functioning.     

Methyllycaconitine and (+)-anatoxin-a differentiate between nicotinic receptors in vertebrate and invertebrate nervous systems

Specific high-affinity binding sites for 125I-alpha-bungarotoxin and (-)-[3H]nicotine have been measured in rat brain and locust (Schistocerca gregaria) ganglia. The binding sites for 125I-alpha-bungarotoxin had similar Kd values of 1.5 x 10(-9) and 0.8 x 10(-9) M for rat and locust preparations, respectively; the corresponding values for the (-)-[3H]nicotine-binding site were 9.3 x 10(-9) and 1.7 x 10(-7) M. Methyllycaconitine (MLA) potently inhibited 125I-alpha-bungarotoxin binding in both rat and locust. MLA was a less effective inhibitor of (-)-[3H]nicotine binding whereas (+)-anatoxin-a was a very potent inhibitor at this site in the rat but not in the locust. These data suggest that (+)-anatoxin-a is a useful probe for the high-affinity nicotine-binding receptor in vertebrate brain, whereas MLA is a preferential probe for the subclass of receptor that binds alpha-bungarotoxin.     

Kinetic Constants for Blood—Brain Barrier Amino Acid Transport in Conscious Rats

The kinetic constants for large neutral amino acid (LNAA) transport across the blood-brain barrier (BBB) of conscious rats were determined in four brain regions: cortex, caudate-putamen, hippocampus, and thalamus-hypothalamus. Indwelling external carotid artery catheters allowed for single-bolus (200 microliters) injections directly into the arterial system of unanesthetized and lightly restrained animals. Our results showed lower brain uptake index values for conscious rats compared to previous reports for anesthetized animals which are consistent with higher rates of cerebral blood flow in the conscious animals. Km values were lower in the conscious animals and ranged from 29% to 87% of the Km values in pentobarbital-anesthetized animals whereas the KD values were about twofold higher in the conscious animals. No apparent regional differences were observed. Influx rates were determined which take into consideration flow rates and plasma amino acid concentrations. Our results showed an average amino acid influx value of 5.2 nmol/min/g, which is 53% higher than the average influx in pentobarbital-anesthetized animals. The present results in conscious animals regarding the low Km of LNAA transport across the BBB lend further support to the importance of fluctuations in plasma amino acid concentrations and LNAA transport competitive effects on brain amino acid availability.     

Blood–Brain Barrier Transport of Valproic Acid

Valproic acid distribution in brain is less than that of other anticonvulsants such as phenytoin or phenobarbital. Possible mechanisms for this decreased distribution space in brain include (a) increased plasma protein binding of valproate relative to the other anticonvulsants and (b) asymmetric blood-brain barrier (BBB) transport of valproate such that the brain-to-blood flux exceeds the blood-to-brain flux. These mechanisms are investigated in the present studies using the intracarotid injection technique in rats and rabbits. In the rat, the brain uptake index (BUI) of [14C]valproate relative to [3H]water is 51 +/- 6%, indicating the blood-to-brain transport of water is twofold greater than that of valproate. However, the BUI of [14C]valproate relative to [3H]water decreased with time after carotid injection during a 4-min washout period, which indicates that brain-to-blood transport of valproate is greater than that of water. This suggests that the permeability of the BBB to valproate is polarized, with antiluminal permeability being much greater than luminal permeability. In rabbits, the BUI of [14C]valproate is 47 +/- 7% in newborns and 17 +/- 6% in adult animals. However, the high drug extraction in newborns may be attributed to decreased cerebral blood flow in the neonate as the BBB permeability-surface area (PS) products are unchanged (e.g., PS = 0.13 and 0.11 ml min-1 X g-1 in the newborn and adult rabbit, respectively). With regard to plasma protein binding effects on valproate transport, brain valproate uptake was also measured in the presence of human, lamb, pig, rat, horse, goat, hamster, dog, and mouse sera. Higher brain uptakes were observed when the unbound fraction of drug increased. However, our data indicate that a fraction of the valproic acid entering the capillaries bound to plasma proteins had the capacity to equilibrate with brain because of enhanced drug dissociation from albumin in the brain microcirculation. Since plasma protein-bound valproate is available for uptake by brain, the major factor underlying the diminished distribution of the drug in brain appears to be the asymmetric transport properties of the BBB to valproic acid.