Neutral Amino Acid Transport System Research Articles

<i>In Vitro</i> Study of L-Glutamate and L-Glutamine Transport in Retinal Pericytes: Involvement of Excitatory Amino Acid Transporter 1 and Alanine–Serine–Cysteine Transporter 2

L-Glutamate (L-Glu) is known to be a relaxant of pericytes and to induce changes in microcirculatory hemodynamics. Since the concentration of L-Glu which induces the dilation of retinal capillaries is reported to be high compared with the estimated concentration in the retinal interstitial fluid, it is hypothesized that some systems involving concentrative L-Glu release are present in retinal pericytes. The purpose of this study was to investigate the existence of L-Glu-storing systems, which contribute to autocrine L-Glu release, in retinal pericytes using conditionally immortalized rat retinal pericytes (TR-rPCT1 cells), which express mRNAs of L-Glu-synthesizing enzymes from L-glutamine (L-Gln). TR-rPCT1 cells express the mRNAs of vesicular L-Glu transporter 1 (VGLUT1), indicating that L-Glu in the cytoplasm is taken up into VGLUT1-expressing vesicles of retinal pericytes. L-Glu and L-Gln are taken up into TR-rPCT1 cells via Na(+)-dependent saturable process(es) with a Km value of 22.4 µM and 163 µM, respectively. The [(3)H]L-Glu uptake was inhibited by ca. 50% in the presence of D-aspartate, a substrate of excitatory amino acid transporter (EAAT) subtypes, whereas substrates of alanine-serine-cysteine transporter (ASCT) subtypes exhibited only a weak inhibitory effect on [(3)H]L-Glu uptake compared with D-aspartate. Regarding the L-Gln uptake by TR-rPCT1 cells, the inhibitory effect of ASCT substrates on the [(3)H]L-Gln uptake was stronger than that of substrates of other neutral amino acid transport systems. Consequently, it was determined that EAAT1 and ASCT2 play a role in the transport of L-Glu and L-Gln, respectively, from retinal interstitial fluid to the cytoplasm of retinal pericytes.

RBE4 cells are highly resistant to paraquat‐induced cytotoxicity: studies on uptake and efflux mechanisms

Paraquat (PQ) is a widely used, highly toxic and non-selective contact herbicide, which has been associated with central neurotoxic effects, namely the development of Parkinson's disease, but whose effects to the blood-brain barrier (BBB) itself have rarely been studied. This work studied the mechanisms of PQ uptake and efflux in a rat's BBB cell model, the RBE4 cells. PQ is believed to enter cells using the basic or neutral amino acid or polyamine transport systems or through the choline-uptake system. In contrast, PQ efflux from cells is reported to be mediated by P-glycoprotein. Therefore, we evaluated PQ-induced cytotoxicity and the effect of some substrates/blockers of these transporters (such as arginine, L-valine, putrescine, hemicholinium-3 and GF120918) on such cytotoxicity. RBE4 cells were shown to be extremely resistant to PQ after 24 h of exposure; even at concentrations as high as 50 mM approximately 45% of the cells remained viable. Prolonging exposure until 48 h elicited significant cytotoxicity only for PQ concentrations above 5 mM. Although hemicholinium-3, a choline-uptake system inhibitor, significantly protected cells against PQ-induced toxicity, none of the effects were observed for arginine, L-valine or putrescine. Meanwhile, inhibiting the efflux pump P-glycoprotein using GF120918 significantly enhanced PQ-induced cytotoxicity. In conclusion, PQ used the choline-uptake system, instead of the transporters for the basic or neutral amino acids or for the polyamines, to enter RBE4 cells. P-glycoprotein extrudes PQ back to the extracellular medium. However, this efflux mechanism only partially explains the observed RBE4 resistance to PQ.

Small Neutral Amino Acid Ester Prodrugs of Acyclovir Targeting Amino Acid Transporters on the Cornea: Possible Antiviral Agents Against Ocular HSV-1 Infections.

The aim of this study was to characterize the affinity and permeability patterns of the amino acid ester prodrugs of acyclovir (ACV), L-alanine-ACV (AACV), L-serine-ACV (SACV), L-serine-succinate-ACV (SSACV) and L-cysteine-ACV (CACV) on rabbit primary corneal epithelial cell culture (rPCEC) and on rabbit cornea. Amino acid prodrugs of acyclovir, AACV, SACV, SSACV and CACV were synthesized in our laboratory. Chemical hydrolysis in aqueous buffer, enzymatic hydrolysis in corneal homogenates and transport across freshly excised rabbit cornea of these prodrugs were studied. SSACV inhibited the uptake of [(3)H] L-alanine on rPCEC and across the intact rabbit cornea. Lineweaver-Burk plot transformation revealed competitive inhibition between L-alanine and SSACV. In corneal tissue homogenate, the half lives of SSACV, SACV and CACV (t1/2) were observed to be 3.5 ± 0.4, 9.2 ± 0.6 and 1.8 ± 0.1 hr respectively, whereas AACV was readily converted to the active parent drug acyclovir exhibiting complete degradation before 5 min. Interestingly translocation of SACV across cornea was inhibited in the presence of 5 mM arginine (~51%), a specific substrate for cationic transport system and in presence of BCH (~38%), a substrate specific for large neutral amino acid transport system (LAT) or cationic and neutral amino acid transport system (B(0,+)). SACV exhibited higher permeability across cornea along with excellent antiviral activity against herpes simplex virus (HSV-1) and varicella-zoster virus (VZV) in comparison to ACV. Recognition by multiple transporters, stability in corneal homogenate and changes in physico-chemical properties contributed to the increased permeability of SACV across cornea.

Molecular and functional identification of large neutral amino acid transporters LAT1 and LAT2 and their pharmacological relevance at the blood-brain barrier.

We present here the evidence of molecular and functional expression of LAT1 and LAT2, subunits of the large neutral amino acid transporter system L, in cultured brain capillary endothelial cells of the rat. By means of the RT-PCR method, transcripts of LAT1, LAT2 and heavy chain of 4F2 antigen (4F2hc) were detected in rat primary cultured brain capillary endothelial cells (BCECs) and immortalized subline, RBEC1. The uptake properties of RBEC1, such as [3H]leucine and L-[3H]DOPA uptake, were similar to those of primary cultured BCECs. So, RBEC1 may retain almost native properties of the large neutral amino acid uptake activities. [3H]Leucine uptake by RBEC1 showed two saturable components and the Km values of the high- and low-affinity components were 8.92+/-3.18 and 119+/-45 microM, respectively. The Km value of the high-affinity component agreed well with that of LAT1 and the amino acid transport selectivity of RBEC1 was similar to that of LAT1. Therefore, it is suggested that LAT1 is important at the blood-brain barrier of rats. Additionally, the Km value of the low-affinity component was similar to that of LAT2. These observations indicate that LAT1 and LAT2 are involved as transporters for large neutral amino acids at the blood-brain barrier. Additionally, we concluded that RBEC1 is useful as an in-vitro model for evaluation of the pharmacological relevance of system L at the blood-brain barrier.

Correlation of l-methyl-11C-methionine (MET) uptake with l-type amino acid transporter 1 in human gliomas

L-type amino acid transporter 1 (LAT1) is a neutral amino acid transport system and is a major route for the transport of large neutral amino acids, including methionine, through the plasma membrane. LAT1 requires the heavy chain of 4F2 cell surface antigen (4F2hc) for its functional expression. Positron emission tomography (PET) with L-[methyl-(11)C] methionine (MET) provides information about amino acid metabolism in brain tumors. We conducted a clinicopathologic study to elucidate the correlation of LAT1 and 4F2hc expression with MET uptake in patients with newly diagnosed human gliomas. Thirty-three newly diagnosed glioma patients were enrolled in this study. Uptake of MET in the tumor was evaluated with the maximum standardized uptake value (SUVmax). Expression of the LAT1, 4F2hc, and CD34, and Ki-67 labeling index of the tumor were analyzed by immunohistochemical staining, and the correlation with the SUVmax in the tumors was examined. Expression of LAT1 and 4F2hc was higher in high-grade gliomas than in low-grade gliomas. The grade of LAT1 immunostaining increased with glioma grade. LAT1 was mainly expressed in the tumor cytoplasm and vascular endothelium and 4F2hc was mainly expressed in the tumor cytoplasm and plasma membrane. Expression of LAT1 but not 4F2hc was significantly correlated with MET SUVmax. Expression of LAT1 in the tumor vascular endothelium is significantly correlated with CD34 positive microvessel density. In conclusion, MET SUVmax correlates with LAT1 expression in the tumor in newly diagnosed gliomas. MET transport may be increased by an increased number of microvessels combined with a higher density or activity of LAT1 in the tumor endothelial cells in high-grade gliomas. Use of MET-PET as a molecular target combined with anti-angiogenesis in glioma therapy should be addressed in future studies.

Small Neutral Amino Acid Ester Prodrugs of Acyclovir Targeting Amino Acid Transporters on the Cornea: Possible Antiviral Agents against Ocular HSV-1 Infections

The aim of this study was to characterize the affinity and permeability patterns of the amino acid ester prodrugs of acyclovir (ACV), L-alanine-ACV (AACV), L-serine-ACV (SACV), L-serine-succinate-ACV (SSACV) and L-cysteine-ACV (CACV) on rabbit primary corneal epithelial cell culture (rPCEC) and on rabbit cornea. Amino acid prodrugs of acyclovir, AACV, SACV, SSACV and CACV were synthesized in our laboratory. Chemical hydrolysis in aqueous buffer, enzymatic hydrolysis in corneal homogenates and transport across freshly excised rabbit cornea of these prodrugs were studied. SSACV inhibited the uptake of [3H] L-alanine on rPCEC and across the intact rabbit cornea. Lineweaver-Burk plot transformation revealed competitive inhibition between L-alanine and SSACV. In corneal tissue homogenate, the half lives of SSACV, SACV and CACV (t1/2) were observed to be 3.5 ± 0.4, 9.2 ± 0.6 and 1.8 ± 0.1 hr respectively, whereas AACV was readily converted to the active parent drug acyclovir exhibiting complete degradation before 5 min. Interestingly translocation of SACV across cornea was inhibited in the presence of 5 mM arginine (~51%), a specific substrate for cationic transport system and in presence of BCH (~38%), a substrate specific for large neutral amino acid transport system (LAT) or cationic and neutral amino acid transport system (B0,+). SACV exhibited higher permeability across cornea along with excellent antiviral activity against herpes simplex virus (HSV-1) and varicella-zoster virus (VZV) in comparison to ACV. Recognition by multiple transporters, stability in corneal homogenate and changes in physico-chemical properties contributed to the increased permeability of SACV across cornea.

The blood-brain barrier in toxicology

The blood-brain barrier in toxicology

Uptake of S-(3-Amino-3-oxopropyl)-cysteine by Caco-2 Cells

Acrylamide is a reactive neurotoxin with a high intestinal bioavailability. Recently we have shown that under the pH regime of the gut acrylamide can react with proteins and that this reaction reduces the uptake of acrylamide in a gut model. On the other hand, using radioactive labeled acrylamide, Bjellaas et al. [Toxicol. Sci. 100, 374-380 (2007)] showed that in vivo the vast majority of orally administered acrylamide is absorbed and excreted as N-acetyl-S-(3-amino-3-oxopropyl)-cysteine with the urine. Therefore, we tested whether intestinal proteases can degrade a protein with acrylamide bound to cysteine residues. Furthermore we tested whether the product of this reaction, S-(3-amino-3-oxopropyl)-cysteine, can pass the intestinal barrier. Here we showed that S-(3-amino-3-oxopropyl)-cysteine is indeed a product of proteolytic degradation of acrylamide-treated proteins. Using Caco-2 cells as a gut model, we further showed that the non-protein amino acid S-(3-amino-3-oxopropyl)-cysteine is a substrate for the neutral and cationic amino acid transporter system. Hence we concluded that protein-bound acrylamide can be released in the intestine and that the resulting product S-(3-amino-3-oxopropyl)-cysteine is transported through the intestinal barrier and later excreted via the urine.

Glutamine metabolism and signaling in the liver

Glutamine is the most abundant amino acid in the human body and can be synthesized by almost all tissues by the glutamine synthetase (GS)-catalyzed amidation of glutamate. Hepatocytes have access to extracellular glutamine by the concentrative uptake via members of the sodium-dependent neutral amino acid transport systems N and A. Hepatic glutamine metabolism in connection with urea synthesis is importantly involved in systemic ammonia detoxication and pH regulation due to the unique regulatory properties of the liver-type glutaminase, the acinar compartimentation of urea and glutamine synthesis, and a cycling of glutamine between periportal and perivenous hepatocytes. Upregulation of GS expression in hepatocellular carcinoma is related to growth advantage and an enhanced metastatic potential. Glutamine is a potent activator of signal transduction. Recent progress concerns the understanding of glutamine-induced hepatocyte swelling and the downstream activation of integrins, Src, and MAP-kinases in the regulation of autophagic proteolysis, canalicular bile acid excretion, glycogen and fatty acid synthesis, insulin signaling, and protection from apoptosis. Most recently the first primary GS defect leading to inherited glutamine deficiency with fatal outcome was described in human. This review summarizes recent progress in the understanding of glutamine metabolism and signal transduction, which provides further rationale for the use of glutamine as a therapeutic tool.

IGF regulation of neutral amino acid transport in the BeWo choriocarcinoma cell line (b30 clone): Evidence for MAP kinase-dependent and MAP kinase-independent mechanisms

Objective IGF-1 and IGF-1 receptors are major determinants of fetal growth and are expressed primarily on the maternal-facing surface of the syncytiotrophoblast cell membrane in the human placenta. IGF-1 regulates fetal growth, in part, by regulating amino acid transport across the placenta. The objective of these studies was to study the role of IGF-1 and its signaling pathway in regulating neutral amino acid transport in a human trophoblast cell culture model. Design The regulation of neutral amino acid transport by IGF-1 was studied in cultured BeWo b30 choriocarcinoma cells using the non-metabolizing amino acid analog, [ 3H]-α-aminoisobutyric acid (AIB). Transport in the absence of Na was used to distinguish system L from total AIB transport. Similarly, Na-dependent transport in the presence of excess methyl-AIB (MeAIB) permitted discrimination of systems A (MeAIB-sensitive) and ASC (MeAIB-insensitive). Specific inhibitors of intracellular signaling pathways were then used to determine the signaling pathway utilized by IGFs to regulate each amino acid transport system. Specificity of inhibition was assessed using specific markers of p70 S6 kinase activity and MAP kinase activation. Results Maximal stimulating concentrations of IGF-I (100 ng/ml) stimulated AIB transport by 30–40% exclusively through system A. Wortmannin (100 nM), an inhibitor of PI-3-kinase activity, inhibited all IGF-I-stimulated transport. Rapamycin (100 ng/ml), an inhibitor of p70 S6 kinase, and bisindolylmaleimide, an inhibitor of protein kinase C (PKC), had no effect. PD-098059 (50 μM), an inhibitor of MAP kinase activation, inhibited 20–30% of basal AIB transport but did not inhibit IGF-I-stimulated transport under the conditions studied. IGF-1 did not increase steady state mRNA levels of the system A transporters, SNAT1 and SNAT2, suggesting IGF-1 stimulates transport via post-transcriptional mechanisms. Conclusions These data demonstrate that IGF-I stimulates neutral amino acid transport system A by a PI3-kinase dependent, post-transcriptional pathway in the BeWo b30 cell line. Additionally, system A activity appear to be sensitive to MAP kinase-dependent pathways not regulated by IGFs.

Functional expression and adaptive regulation of Na +-dependent neutral amino acid transporter SNAT2/ATA2 in normal human astrocytes under amino acid starved condition

We reported here the functional characteristics of Na +-dependent neutral amino acid transport system A in normal human astrocytes and its adaptive regulation, a process in which amino acid starvation induces the transport activity. Reverse transcription-PCR revealed that the system A transporter subtype, SNAT2/ATA2, is only expressed in these cells. The other two known system A transporter subtypes, SNAT1/ATA1 and SNAT4/ATA3, could not be detected. Na +-dependent uptake of α-(methylamino)isobutyric acid, a specific model substrate for system A, was pH-sensitive and saturable with a Michaelis–Menten constant of 0.22 ± 0.03 mM. Exposures of human astrocytes to amino acid-free medium increased the system A activity and the steady-state levels of SNAT2/ATA2 mRNA in an exposure time-dependent manner. This stimulatory effect was attenuated significantly by actinomycin D, an inhibitor of RNA synthesis, and cycloheximide, an inhibitor of protein synthesis. Taken collectively, these data show that chronic exposure (6 h) of the cells to the amino acid-free medium increases the system A activity most likely by enhancing de novo synthesis of the transporter protein and consequently increasing the density of the transporter protein in the plasma membrane.

Influence of Dietary Protein Levels on the Fate of Methylmercury and on Amino Acid Transport at the Renal Brush Border Membrane in Rats

We investigated the influence of dietary protein levels on the fate of methylmercury (MeHg) in rats, and the difference in its urinary excretion was discussed from the viewpoint of the dietary protein level-dependent alteration in activity of the neutral amino acid transport system, through which MeHg metabolites such as MeHg-L-cysteine are reabsorbed, at the renal brush border membrane. When MeHg was administered to rats fed either a 24.8% protein diet (normal protein diet, NPD) or a 7.5% protein diet (low protein diet, LPD), urinary Hg excretion was much lower in LPD-fed rats than in NPD-fed rats, whereas no difference was observed in fecal excretion 1 day after MeHg administration. At that time, Hg concentrations in the brain and plasma were similar in the two dietary groups, whereas the concentrations in liver and blood were higher, but the renal concentration was lower in LPD-fed rats than in NPD-fed rats. Regardless of the presence of Na+, uptake of 14C-L-phenylalanine for the first 20 sec was higher in the renal brush border vehicles from LPD-fed rats than in those from NPD-fed rats. These results suggest that dietary protein deficiency enhances the neutral amino acid transport at the renal brush border membrane, which could lead efficient reabsorption of MeHg metabolites from the proximal luminal space, and it might play a key role in the marked decrease in urinary excretion of MeHg.

Transcriptional Control of the Human Sodium-coupled Neutral Amino Acid Transporter System A Gene by Amino Acid Availability Is Mediated by an Intronic Element

System A amino acid transporter (SNAT2) gene expression is up-regulated at the transcriptional level in response to amino acid deprivation. Functional analysis of genomic fragments 5' upstream of the transcription start site, for both human and mouse SNAT2 genes showed that these regions exhibit promoter activity, but were amino acid unresponsive. However, when the human and mouse constructs were extended to include intron 1, it was observed that the rate of transcription was increased following amino acid deprivation. Deletion analysis of the human gene identified an intron 1 sequence spanning 54 nucleotides that was sufficient for conferring amino acid-dependent regulation to a minimal SNAT2 promoter. Alignment of the corresponding region from the human, mouse, and rat genomes revealed three highly conserved sequences. From site-directed mutagenesis, it was concluded that one of these sites functions as an amino acid response element (AARE) to regulate transcription. The core sequence of this site is identical to the AARE in the human CHOP gene. The SNAT2 AARE, along with a nearby conserved CAAT box, has enhancer activity in that it functions in an orientation and position independent manner, and it confers regulated transcription to a heterologous promoter.

Nitric oxide synthesis requires activity of the cationic and neutral amino acid transport system y+L in human umbilical vein endothelium.

L-arginine transport is mediated by the cationic/neutral amino acid transport system y+L and cationic amino acid transporters y+/CATs in human umbilical vein endothelial cells (HUVECs). System y+/CATs activity may be rate-limiting for nitric oxide (NO) synthesis, but no reports have demonstrated system y+L involvement in NO synthesis in endothelium. We investigated the role of system y+L in NO synthesis in HUVECs. Transport of 1.5 microM L-arginine was inhibited (P < 0.05) by L-lysine (K(i), 1.4 micro M), L-leucine (K(i), 1.8 micro M) and L-phenylalanine (K(i), 4.1 microM), but was unaltered (P > 0.05) by L-alanine or L-cysteine. The system y+/CATs inhibitor, N-ethylmaleimide (NEM), did not alter 1.5 microM L-arginine transport, but inhibited (92 +/- 3 %) 100 microM L-arginine transport. L-arginine transport in the presence of NEM was saturable (V(max), 0.37 +/- 0.02 pmol (microg protein)(-1) min(-1); K(m), 1.5 +/- 0.3 microM) and competitively inhibited by L-leucine in the presence of Na+ (V(max), 0.49 +/- 0.06 pmol (microg protein)(-1) min(-1); K(m), 6.5 +/- 0.9 microM). HUVECs express SLC3A2/4F2hc, SLC7A7/4F2-lc2 and SLC7A6/4F2-lc3 genes encoding for the high-affinity transport system y+L. N(G)-Nitro-L-arginine methyl ester and L-leucine, but not NEM, inhibited NO synthesis in medium containing 1.5 microM L-arginine. Cells exposed to 25 mM D-glucose (24 h) exhibited reduced system y+L activity (V(max), 0.15 +/- 0.008 pmol (microg protein)(-1) min(-1); K(m), 1.4 +/- 0.3 microM) and NO synthesis. However, 25 mM D-glucose increased NO synthesis and L-arginine transport via system y+. Thus, L-arginine transport through system y+L plays a role in NO synthesis, which could be a determining factor in pathological conditions where the endothelial L-arginine-NO pathway is altered, such as in diabetes mellitus.

Uptake of [ 3H] l-serine in rat brain synaptosomal fractions

Accumulation of [ 3H] l-serine in crude synaptosomal fractions freshly prepared from rat brain has been found to be temperature-sensitive and to consist of both Na +-dependent and Na +-independent components. The accumulation of [ 3H] l-serine measured at submicromolar concentrations had a distinct substrate selectivity, different from the uptake of [ 3H] l-proline, [ 3H] l-glutamate and [ 3H]GABA. It was fully inhibited by l-glutamine, l-asparagine, l-cysteine, l-alanine, l-leucine, l-isoleucine, l-tyrosine, l-phenylalanine, l-threonine and by the synthetic marker for the large neutral amino acid transport systems 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid, but not influenced by β-alanine, taurine, glycine nor was it inhibited by the marker for the A system, l-2-methylamino isobutyric acid. d-Serine at 1 mM concentration produced no significant inhibition of the accumulation of 10 nM [ 3H] l-serine. We conclude that l-serine uptake observed in the present study is mediated by at least two distinct transport systems: a Na +-dependent one of lower affinity ( K m in mM range) and a Na +-independent system of higher affinity ( K m≈20–100 μM). Characteristics of [ 3H] l-serine accumulation displayed at low substrate concentrations suggest that it was mediated neither by the typical ‘A’, nor by the ‘large neutral’, amino acid transport systems but predominantly by transporters belonging to the recently identified LAT ( l-amino acid transporter) family.

Effects of L-dopa and other amino acids against paraquat-induced nigrostriatal degeneration.

Exposure to the herbicide paraquat causes selective nigrostriatal degeneration and aggregation of alpha-synuclein in the mouse brain. The purpose of this study was to assess mechanisms of paraquat entry into the CNS and, in particular, the effects of substrates of the blood-brain barrier (BBB) neutral amino acid transporter (System L carrier) on paraquat accumulation and neurotoxicity. Using a paraquat antibody, robust immunoreactivity was observed in the midbrain of mice injected with the herbicide. This immunoreactivity was abolished by administration of l-valine or l-phenylalanine, two System L substrates, immediately before paraquat exposure. Pre-treatment with these amino acids completely protected against paraquat-induced loss of nigrostriatal dopaminergic cells and formation of thioflavine S-positive intracellular deposits. Interestingly, the anti-parkinsonian drug l-dopa, which is transported across the BBB through the same neutral amino acid carrier, was also neuroprotective when administered 30 min prior to paraquat. In contrast, paraquat-induced toxicity was unaffected if animals (i) were pre-treated with d-valine, the biologically inactive d-isomer of l-valine, or with l-lysine, a substrate of the basic rather than the neutral amino acid carrier, or (ii) were injected with l-dopa 24 h after paraquat exposure. Data are consistent with a critical role of uptake across the BBB in paraquat neurotoxicity, and suggest that dietary elements (e.g. amino acids) or therapeutic agents (e.g. l-dopa) may modify the effects of toxicants targeting the nigrostriatal system.

Molecular mechanisms of drug influx and efflux transport at the blood-brain barrier

The blood-brain barrier (BBB) segregates the circulating blood from interstitial fluid in the brain and restricts drug permeability into the brain. Recent studies have revealed that the BBB exhibits not only blood-to-brain influx transport for the supply of nutrients, but also brain-to-blood efflux transport to excrete drugs and endogenous compounds. The influx transport system allows drugs to enter the brain. (L)-DOPA is transported into the brain by the large neutral amino acid transport system, system L. A cationic mu-opioid peptide analogue enters the brain by adsorptive-mediated endocytosis. In contrast, efflux transport limits the distribution of drugs in the brain. The ATP binding cassette transporter B1 (ABCB1) mediates the efflux transport of lipophilic drugs at the BBB by using ATP energy. Furthermore, organic anion transporter 3 (OAT3) is expressed at the BBB and mediates the efflux transport of homovanillic acid, a dopamine metabolite. This efflux transport is also likely to be involved in the transport of anionic drugs such as 6-mercaptopurine and acyclovir. Clarifying the BBB transport could give us important information allowing the development of better CNS drugs and improving our understanding of the relationship between CNS diseases and BBB functions.

Pralidoxime iodide (2-pAM) penetrates across the blood-brain barrier.

The in vivo rat brain microdialysis technique with HPLC/UV was used to determine the blood-brain barrier (BBB) penetration of pralidoxime iodide (2-PAM), which is a component of the current nerve agent antidote therapy. After intravenous dosage of 2-PAM (10, 50, 100 mg/kg), 2-PAM appeared dose-dependently in the dialysate; the striatal extracellular/blood concentration ratio at 1 h after 50 mg/kg dosage was 0.093 +/- 0.053 (mean +/- SEM). This finding offered conclusive evidence of the BBB penetration of 2-PAM. We also examined whether the BBB penetration of 2-PAM was mediated by a certain specific transporter, such as a neutral or basic amino acid transport system. Although it was unclear, the neural uptake of 2-PAM was Na+ dependent. The mean BBB penetration by 2-PAM was approximately 10%, indicating the intravenous administration of 2-PAM might be to a degree effective to reactivation of the blocked cholinesterase in the brain.

Carrier mediated uptake of l-tyrosine and its competitive inhibition by model tyrosine linked compounds in a rabbit corneal cell line (SIRC)—strategy for the design of transporter/receptor targeted prodrugs

The objective of this study was to investigate the presence of amino acid transporters on the corneal epithelium and to enhance corneal drug absorption through prodrug modification targeted to the amino acid transporters. SIRC was used as a model cell line representing the corneal epithelium. Uptake studies were carried out using [ 3H] l-tyrosine at 37 °C. Temperature, energy and pH dependence studies were carried out. The uptake seems to be composed of a major saturable and minor non-saturable component ( V max=2.9±0.62 nmoles/min/mg protein, K m=71±21 μM, K d=2.6±0.6 nl/min/mg protein). No significant inhibition of uptake was observed in the presence of metabolic inhibitors or in the absence of sodium. Competitive inhibition studies were performed in the presence of various amino acids and model tyrosine conjugates ( p-nitro and p-chloro benzyl ether conjugate of l-tyrosine). Uptake was inhibited by neutral aromatic and large neutral aliphatic amino acids. l-Tyrosine uptake was inhibited by its ether conjugates in a concentration dependent manner suggesting that these compounds may be sharing the same transport mechanism. This study provides biochemical evidence of the presence of a large neutral amino acid transport system on the corneal epithelium, which may be utilized to enhance the corneal drug transport.

Effects of different oxidizing agents on neutral amino acid transport systems in isolated bovine brain microvessels

Using isolated bovine brain microvessels as an in vitro model of the blood–brain barrier (BBB) we have evaluated the role of free radical generating solutions on some amino acid transport systems operating on the endothelial cell membrane. Fe 2+/ascorbate, phenylhydrazine and CuSO 4 did not affect any of the transport system tested, while exposure of bovine brain microvessels to tert-butylhydroperoxide ( t-BHP) caused a reduced capacity to take up small neutral amino acids via the Na +-dependent A-system. The presence of glucose during t-BHP treatment did not prevent this inhibition, which was partially counteracted when the isolated microvessels were incubated with 5 mM inosine before the oxidative stress. Incubation of the isolated capillaries with 5 mM dithiothreitol, after exposure to t-BHP, resulted in a 50% recovery of the α-methylaminoisobutyrate (MeAIB) uptake by the A-system. Treatment with t-BHP, which had no effect on the l-system of neutral amino acid transport, caused a significant decrease of the intracellular levels of ATP, of glutathione (GSH), and of γ-glutamyltranspeptidase (GGT) activity, while no significant modification of hexokinase (HK) or of alkaline phosphatase (ALKP) activities were observed. Oxidative damage of the BBB appears therefore to impair essentially the metabolic pathways which ensure the energy requirement for the endothelial cells, thus inhibiting the energy-dependent amino acid transport system “A”.