Targeting the cerebrovascular large neutral amino acid transporter (LAT1) isoform using a novel disulfide-based brain drug delivery system

We describe a novel strategy to achieve high affinity recognition for the specific, cerebrovascular large neutral amino acid transporter (LAT1) isoform by covalent coupling of small molecules to the amino acid, L-cysteine (L-Cys). L-Cys (as the carrier) was covalently attached via a disulfide bond to either 6-mercaptopurine or 2-methyl-1-propanethiol (IBM) to form the brain-targeted drug delivery systems (BTDS). BTDS were designed for high affinity recognition by LAT1 at the cerebrovasculature. Using an in situ rat brain perfusion technique, competition between BTDS and the radiotracer [¹⁴C]L-Leu demonstrated significant inhibition of [¹⁴C]L-Leu brain uptake. BTDS possess affinity for cerebrovascular LAT1 in many distinct brain compartments, and the recognition of BTDS by LAT1 is influenced by hydrophobicity of the side-chain in BTDS. Thus, the BTDS strategy may be utilized for rapid shuttling of various neuropharmaceuticals into brain.     

Involvement of LAT1 and LAT2 in the high- and low-affinity transport of L-leucine in human retinal pigment epithelial cells (ARPE-19 cells)

System L, which is encoded by LAT1 and LAT2, is an amino acid transport system that transports neutral amino acids, including several essential amino acids in an Na⁺-independent manner. Due to its broad substrate selectivity, system L has been proposed to mediate the transport of amino-acid-related drugs across the blood–tissue barriers. We characterized L-leucine transport and its corresponding transporter in a human retinal pigment epithelial cell line (ARPE-19 cells) as an in vitro model of the outer blood–retinal barrier. [³H]L-leucine uptake by ARPE-19 cells took place in an Na⁺-, Cl^?-independent and saturable manner with K_m values of 8.71 and 220 µM. This process was more potently cis-inhibited by substrates of LAT1 than those of LAT2. [³H]L-leucine efflux from ARPE-19 cells was trans-stimulated by substrates of LAT1 and LAT2 through the obligatory exchange mechanism of system L. Although RT-PCR analysis demonstrated that LAT1 and LAT2 mRNA are expressed in ARPE-19 cells, the LAT1 mRNA concentration is 42-fold higher than that of LAT2. Moreover, immunoblot analysis demonstrated that LAT1 is expressed in ARPE-19 cells. In conclusion, although the transport function of LAT1 is greater than that of LAT2, LAT1 and LAT2 are involved in L-leucine transport in ARPE-19 cells. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 2475–2482, 2010     

Reabsorption of neutral amino acids mediated by amino acid transporter LAT2 and TAT1 in the basolateral membrane of proximal tubule

In order to understand the renal reabsorption mechanism of neutral amino acids via amino acid transporters, we have isolated human L-type amino acid transporter 2 (hLAT2) and human T-type amino acid transporter 1 (hTAT1) in human, then, we have examined and compared the gene structures, the functional characterizations and the localization in human kidney. Northern blot analysis showed that hLAT2 mRNA was expressed at high levels in the heart, brain, placenta, kidney, spleen, prostate, testis, ovary, lymph node and the fetal liver. The hTAT1 mRNA was detected at high levels in the heart, placenta, liver, skeletal muscle, kidney, pancreas, spleen, thymus and prostate. Immunohistochemical analysis on the human kidney revealed that the hLAT2 and hTAT1 proteins coexist in the basolateral membrane of the renal proximal tubules. The hLAT2 transports all neutral amino acids and hTAT1 transports aromatic amino acids. The basolateral location of the hLAT2 and hTAT1 proteins in the renal proximal tubule as well as the amino acid transport activity of hLAT2 and hTAT1 suggests that these transporters contribute to the renal reabsorption of neutral and aromatic amino acids in the basolateral domain of epithelial proximal tubule cells, respectively. Therefore, LAT2 and TAT1 play essential roles in the reabsorption of neutral amino acids from the epithelial cells to the blood stream in the kidney. Because LAT2 and TAT1 are essential to the efficient absorption of neutral amino acids from the kidney, their defects might be involved in the pathogenesis of disorders caused by a disruption in amino acid absorption such as blue diaper syndrome.     

Evaluation of amino acid-mustard transport as L-type amino acid transporter 1 (LAT1)-mediated alkylating agents

The L-type amino acid transporter 1 (LAT1, SLC7A5) is an Na+-independent neutral amino acid transporter the expression of which is located in retinal endothelial cells. Due to its broad substrate selectivity, LAT1 has been proposed to mediate the transport of amino acid-related drugs across the blood-tissue barriers. Here, we have investigated the transport screening of amino acid-mustards using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) which expresses LAT1. We synthesized 5 amino acid-mustards: tyrosine-mustard, phenylglycine-mustard, alanine-mustard, ornithine-mustard, and lysine-mustard. LAT1-mediated [3H]L-phenylalanine (Phe) uptake by TR-iBRB2 cells was inhibited in a competitive manner by tyrosine-mustard and phenylglycine-mustard as well as melphalan (phenylalanine-mustard). Phenylglycine-mustard was able to induce the efflux of [3H]Phe preloaded into the TR-iBRB2 cells expressing LAT1 through the obligatory exchange mechanism, although tyrosine-mustard, alanine-mustard, ornithine-mustard, lysine-mustard, and melphalan did not induce any significant efflux. These findings suggest that phenylglycine-mustard is a better substrate for LAT1 than melphalan and other amino acid-mustards.     

D-cycloserine transport in human intestinal epithelial (Caco-2) cells: mediation by a H(+)-coupled amino acid transporter.

1. The ability of D-cycloserine to act as a substrate for H+/amino acid symport has been tested in epithelial layers of Caco-2 human intestinal cells. 2. In Na(+)-free media with the apical bathing media held at pH 6.0, D-cycloserine (20 mM) is an effective inhibitor of net transepithelial transport (Jnet) of L-alanine (100 microM) and its accumulation (across the apical membrane) in a similar manner to amino acid substrates (L-alanine, beta-alanine, L-proline and glycine). In contrast L-valine was ineffective as an inhibitor for H+/amino acid symport. Both inhibition of L-alanine Jnet and its accumulation by D-cycloserine were dose-dependent, maximal inhibition being achieved by 5-10 mM. 3. Both D-cycloserine and known substrates for H+/amino acid symport stimulated an inward short circuit current (Isc) when voltage-clamped monolayers of Caco-2 epithelia, mounted in Ussing chambers, were exposed to apical substrate in Na(+)-free media, with apical pH held at 6.0. The D-cycloserine dependent increase in Isc was dose-dependent with an apparent Km = 15.8 +/- 2.0 (mean +/- s.e. mean) mM, and Vmax = 373 +/- 21 nmol cm-2h-1. 4. D-Cycloserine (20 mM) induced a prompt acidification of Caco-2 cell cytosol when superfused at the apical surface in both Na+ and Na(+)-free conditions. Cytosolic acidification in response to D-cycloserine was dependent upon superfusate pH, being attenuated at pH 8 and enhanced in acidic media. 5. The increment in Isc with 20 mM D-cycloserine was non-additive with other amino acid substrates for H+/amino acid symport.     

Pharmacokinetic role of L-type amino acid transporters LAT1 and LAT2

LAT1 and LAT2 are heterodimeric large amino acid transporters that are expressed in various tissues, including the intestinal wall, blood-brain barrier, and kidney. These transporters consist of membrane spanning light chain and heavy chain, and they act as 1:1 exchangers in concert with other amino acid transporters. Only a few drugs (less than 10) are substrates of LAT1 and LAT2, including l-DOPA, alpha-methyldopa, melphalan, and gabapentin. The mechanisms and substrates have been mostly elucidated using mammalian cells and Xenopus oocytes. The in vivo relevance of LAT1 and LAT2 in pharmacokinetics is obscure, because contradictory findings have been reported. It is difficult to make quantitative pharmacokinetic conclusions about LAT1 and LAT2. This is due to the possible involvement of other transporters (including cross-linked heterodimers of light chain with different heavy chains, other overlapping transporters, for example TAT1), competing endogenous amino acids, and saturation phenomena. This review presents the current functional knowledge on LAT1 and LAT2 with emphasis on their potential involvement in pharmacokinetics.     

Functional characterization of monocarboxylic acid,large neutral amino acid,bile acid and peptide transporters,and P-glycoprotein in MDCK and Caco-2 cells

Bidirectional transport studies were conducted to determine whether substrates of five intestinal transporters showed carrier-mediated asymmetric transport across MDCK (Madin-Darby canine kidney) cell monolayers grown under standard conditions. Drug concentrations were quantitated using liquid scintillation counting, liquid chromatography/mass spectrometry/mass spectrometry, or liquid chromatography/mass spectrometry. In the presence of a pH gradient, benzoic acid exhibited net apical-to-basolateral transport, with apparent permeability ratios (apical-to-basolateral permeability/basolateral-to-apical permeability) ranging from 14 to 25. The addition of valproic acid reduced the permeability ratio by 70-90%. Cephalexin transport also exhibited net absorption in the presence of a pH gradient, with apparent permeability ratios ranging from 14 to 71, depending on growth conditions. Radiolabeled phenylalanine exhibited a low level of carrier-mediated absorption with an apparent permeability ratio of 1.8 that was reduced to 1.0 in the presence of unlabeled L-phenylalanine. Taurocholic acid did not exhibit carrier-mediated absorption. Cyclosporine and fexofenadine exhibited P-glycoprotein-mediated efflux from both MDCK and Caco-2 cells, which was more sensitive to inhibition in MDCK cells. These results suggest that although MDCK cell monolayers may be a useful model for evaluating transport by the absorptive monocarboxylic acid and peptide transporters and the efflux transporter, P-glycoprotein, they are not useful for predicting large neutral amino acid or bile acid transport in the intestine.     

Substrate specificity and functional characterisation of the H+/amino acid transporter rat PAT2 (Slc36a2)

Functional characteristics and substrate specificity of the rat proton-coupled amino acid transporter 2 (rat PAT2 (rPAT2)) were determined following expression in Xenopus laevis oocytes using radiolabelled uptake measurements, competition experiments and measurements of substrate-evoked current using the two-electrode voltage-clamp technique. The aim of the investigation was to determine the structural requirements and structural limitations of potential substrates for rPAT2.Amino (and imino) acid transport via rPAT2 was pH-dependent, Na(+)-independent and electrogenic. At extracellular pH 5.5 (in Na(+)-free conditions) proline uptake was saturable (Km 172+/-41 muM), demonstrating that rPAT2 is, relative to PAT1, a high-affinity transporter.PAT2 preferred substrates are L-alpha-amino acids with small aliphatic side chains (e.g. the methyl group in alanine) and 4- or 5-membered heterocyclic amino and imino acids such as 2-azetidine-carboxylate, proline and cycloserine, where both D- and L-enantiomers are transported.The major restrictions on transport are side chain size (the ethyl group of alpha-aminobutyric acid is too large) and backbone length, where the separation of the carboxyl and amino groups by only two CH(2) groups, as in beta-alanine, is enough to reduce transport. Methylation of the amino group is tolerated (e.g. sarcosine) but increasing methylation, as in betaine, decreases transport. A free carboxyl group is preferred as O-methyl esters show either reduced transport (alanine-O-methyl ester) or are excluded.The structural characteristics that determine the substrate specificity of rPAT2 have been identified. This information should prove valuable in the design of selective substrates/inhibitors for PAT1 and PAT2.     

Molecular and functional characterization of the human platelet Na(+) /Ca(2+) exchangers

BACKGROUND AND PURPOSE The Na(+) /Ca(2+) exchanger is a bi-directional transporter that plays an important role in maintaining the concentration of cytosolic Ca(2+) ([Ca(2+) ](i) ) of quiescent platelets and increasing it during activation with some, but not all, agonists. There are two classes of Na(+) /Ca(2+) exchangers: K(+) -independent Na(+) /Ca(2+) exchanger (NCX) and K(+) -dependent Na(+) /Ca(2+) exchanger (NCKX). Platelets have previously been shown to express NCKX1. However, initial studies from our laboratory suggest that NCX may also play a role in platelet activation. The objective of this study was to determine if the human platelet expresses functional NCXs. EXPERIMENTAL APPROACH RT-PCR, DNA sequencing and Western blot analysis were utilized to characterize the human platelet Na(+) /Ca(2+) exchangers. Their function during quiescence and collagen-induced activation was determined by measuring [Ca(2+) ](i) with calcium-green/fura-red in response to: changes in the Na(+) and K(+) gradient, NCX pharmacological inhibitors (CBDMB, KB-R7943 and SEA0400) and antibodies specific to extracellular epitopes of the exchangers. KEY RESULTS Human platelets express NCX1.3, NCX3.2 and NCX3.4. The NCXs operate in the Ca(2+) efflux mode in resting platelets and also during their activation with thrombin but not collagen. Collagen-induced increase in [Ca(2+) ](i) was reduced with the pharmacological inhibitors of NCX (CBDMB, KB-R7943 or SEA0400), anti-NCX1 and anti-NCX3. In contrast, anti-NCKX1 enhanced the collagen-induced increase in [Ca(2+) ](i) . CONCLUSIONS AND IMPLICATIONS Human platelets express K(+) -independent Na(+) /Ca(2+) exchangers NCX1.3, NCX3.2 and NCX3.4. During collagen activation, NCX1 and NCX3 transiently reverse to promote Ca(2+) influx, whereas NCKX1 continues to operate in the Ca(2+) efflux mode to reduce [Ca(2+) ](i) .     

Transport of amino acid-related compounds mediated by L-type amino acid transporter 1 (LAT1): insights into the mechanisms of substrate recognition

The L-type amino acid transporter 1 (LAT1) is an Na(+)-independent neutral amino acid transporter subserving the amino acid transport system L. Because of its broad substrate selectivity, system L has been proposed to be responsible for the permeation of amino acid-related drugs through the plasma membrane. To understand the mechanisms of substrate recognition, we have examined the LAT1-mediated transport using a Xenopus laevis oocyte expression system. LAT1-mediated [(14)C]phenylalanine uptake was strongly inhibited in a competitive manner by aromatic-amino acid derivatives including L-dopa, alpha-methyldopa, melphalan, triiodothyronine, and thyroxine, whereas phenylalanine methyl ester, N-methyl phenylalanine, dopamine, tyramine, carbidopa, and droxidopa did not inhibit [(14)C]phenylalanine uptake. Gabapentin, a gamma-amino acid, also exerted a competitive inhibition on LAT1-mediated [(14)C]phenylalanine uptake. Although most of the compounds that inhibited LAT1-mediated uptake were able to induce the efflux of [(14)C]phenylalanine preloaded to the oocytes expressing LAT1 through the obligatory exchange mechanism, melphalan, triiodothyronine, and thyroxine did not induce the significant efflux. Based on the experimental and semiempirical computational analyses, it is proposed that, for an aromatic amino acid to be a LAT1 substrate, it must have a free carboxyl and an amino group. The carbonyl oxygen closer to the amino group needs a computed charge of -0.55 approximately -0.56 and must not participate in hydrogen bonding. In addition, the hydrophobic interaction between the substrate side chain and the substrate binding site of LAT1 seems to be crucial for the substrate binding. A substrate, however, becomes a blocker once Connolly accessible areas become large and/or the molecule has a high calculated logP value, such as those for melphalan, triiodothyronine, and thyroxine.     

Identification and functional characterization of alpha(2)-adrenoceptor polymorphisms.

For each alpha(2)-adrenoceptor subtype (alpha(2A), alpha(2B) and alpha(2C)), sequence variations within the coding region of each gene have been identified in humans. These result in substitutions or deletions of amino acids in the third intracellular loops of each receptor. This article summarizes the genetics and molecular biology of alpha(2)-adrenoceptor polymorphisms, including the consequences of each polymorphism on receptor signaling, as determined in transfected cells. These effects include alterations in G-protein coupling, desensitization and G-protein receptor kinase-mediated phosphorylation. Studies so far provide the mechanistic basis for future studies to investigate genetic risk factors and pharmacogenetics in pathophysiological conditions linked to alpha(2)-adrenoceptor function.     

Impact of system L amino acid transporter 1 (LAT1) on proliferation of human ovarian cancer cells: A possible target for combination therapy with anti-proliferative aminopeptidase inhibitors

Amino acids activate nutrient signaling via the mammalian target of rapamycin (mTOR), we therefore evaluated the relationship between amino acid transporter gene expression and proliferation in human ovarian cancer cell lines. Expression of three cancer-associated amino acid transporter genes, LAT1, ASCT2 and SN2, was measured by qRT-PCR and Western blot. The effects of silencing the LAT1 gene and its inhibitor BCH on cell growth were evaluated by means of cell proliferation and colony formation assays. The system L amino acid transporter LAT1 was up-regulated in human ovarian cancer SKOV3, IGROV1, A2780, and OVCAR3 cells, compared to normal ovarian epithelial IOSE397 cells, whereas ASCT2 and SN2 were not. BCH reduced phosphorylation of p70S6K, a down-stream effector of mTOR, in SKOV3 and IGROV1 cells, and decreased their proliferation by 30% and 28%, respectively. Although proliferation of SKOV3 (S1) or IGROV1 (I10) cells was unaffected by LAT1-knockdown, plating efficiency in colony formation assays was significantly reduced in SKOV3(S1) and IGROV1(I10) cells to 21% and 52% of the respective plasmid transfected control cells, SKOV3(SC) and IGROV(IC), suggesting that LAT1 affects anchorage-independent cell proliferation. Finally, BCH caused 10.5- and 4.3-fold decrease in the IC₅₀ value of bestatin, an anti-proliferative aminopeptidase inhibitor, in IGROV1 and A2780 cells, respectively, suggesting that the combined therapy is synergistic. Our findings indicate that LAT1 expression is increased in human ovarian cancer cell lines; LAT1 may be a target for combination therapy with anti-proliferative aminopeptidase inhibitors to combat ovarian cancer.     

A study of the substrate specificity of Na+-dependent and Na+-independent neutral amino acid transport systems in dog intestinal brush-border membrane vesicles using L-alanine analogues

Neutral amino acids are mainly transported across the intestinal brush-border membrane by two Na+-dependent systems (system B0 and system B0+) and one Na+-independent system (system b0,+). To investigate potential differences in substrate specificity between these systems, we screened ten different alanine analogues for their ability to inhibit the transport of L-alanine in dog intestinal brush-border membrane vesicles. The results suggested that a phenyl group directly attached to the alpha-carbon has different effects on the Na+-dependent and Na+-independent transport systems, with an increased affinity for the former and a decreased affinity for the latter. Based on these inhibition studies, we investigated [14C]L-phenylglycine transport kinetics in comparison with L-alanine. Similar to L-alanine, L-phenylglycine transport followed at least three routes, however, the Km of the Na+-dependent transport system was lower and the K'm of the Na+-independent system was higher than the corresponding values for L-alanine. These results corroborated the conclusions drawn from the inhibition studies. Based on these data, we conclude that different sizes of immediate parts to the alpha-carbon in functional groups of amino acid analogues have differential effects on the interaction of these amino acid analogues with the Na+-dependent and Na+-independent transport systems for neutral amino acids.     

AMP-activated protein kinase (AMPK)-dependent and -independent pathways regulate hypoxic inhibition of transepithelial Na(+) transport across human airway epithelial cells

BACKGROUND AND PURPOSE

Pulmonary transepithelial Na⁺ transport is reduced by hypoxia, but in the airway the regulatory mechanisms remain unclear. We investigated the role of AMPK and ROS in the hypoxic regulation of apical amiloride-sensitive Na⁺ channels and basolateral Na⁺K⁺ ATPase activity.

EXPERIMENTAL APPROACH

H441 human airway epithelial cells were used to examine the effects of hypoxia on Na⁺ transport, AMP : ATP ratio and AMPK activity. Lentiviral constructs were used to modify cellular AMPK abundance and activity; pharmacological agents were used to modify cellular ROS.

KEY RESULTS

AMPK was activated by exposure to 3% or 0.2% O₂ for 60 min in cells grown in submerged culture or when fluid (0.1 mL·cm⁻²) was added to the apical surface of cells grown at the air–liquid interface. Only 0.2% O₂ activated AMPK in cells grown at the air–liquid interface. AMPK activation was associated with elevation of cellular AMP : ATP ratio and activity of the upstream kinase LKB1. Hypoxia inhibited basolateral ouabain-sensitive I_sc (I_ouabain) and apical amiloride-sensitive Na⁺ conductance (G_Na+). Modification of AMPK activity prevented the effect of hypoxia on I_ouabain (Na⁺K⁺ ATPase) but not apical G_Na+. Scavenging of superoxide and inhibition of NADPH oxidase prevented the effect of hypoxia on apical G_Na+ (epithelial Na⁺ channels).

CONCLUSIONS AND IMPLICATIONS

Hypoxia activates AMPK-dependent and -independent pathways in airway epithelial cells. Importantly, these pathways differentially regulate apical Na⁺ channels and basolateral Na⁺K⁺ ATPase activity to decrease transepithelial Na⁺ transport. Luminal fluid potentiated the effect of hypoxia and activated AMPK, which could have important consequences in lung disease conditions.     

Expression and functional characterization of human ABC transporter ABCG2 variants in insect cells

Hitherto three variant forms of ABCG2 have been documented on the basis of their amino acid moieties (i.e., Arg, Gly, and Thr) at the position 482. In the present study, we have generated those variants of ABCG2 by site-directed mutagenesis and expressed them in Sf9 insect cells. The apparent molecular weight of the expressed ABCG2 variants was 130,000 under non-reductive conditions, whereas it was reduced to 65, 000 by treatment with mercaptoethanol. It is suggested that ABCG2 exists in the plasma membrane of Sf9 cells as a homodimer bound through cysteinyl disulfide bond(s). Both ATPase activity and drug transport of ABCG2 variants were examined by using plasma membrane fractions prepared from ABCG2-overexpressing Sf9 cells. The ATPase activity of the plasma membrane expressing ABCG2 (Gly-482) was significantly enhanced by prazosin. In contrast, ABCG2 (Arg-482) transports [(3)H]methotrexate in an ATP-dependent manner; however, no transport activity was observed with the other variants (Gly-482 and Thr-482). It is strongly suggested that the amino acid moiety at the position of 482 is critical for the substrate specificity of ABCG2.     

Identification and functional characterization of new potentially defective alleles of human CYP2C19

CYP2C19 is a clinically important enzyme responsible for the metabolism of a number of therapeutic drugs, such as mephenytoin, omeprazole, diazepam, proguanil, propranolol and certain antidepressants. Genetic polymorphisms in this enzyme result in poor metabolizers of these drugs. There are racial differences in the incidence of the poor metabolizer trait, which represents 13-23% of Asians but only 3-5% of Caucasians. In this study, single nucleotide polymorphisms (SNPs) in CYP2C19 were identified by direct sequencing of genomic DNA from 92 individuals from three different racial groups of varied ethnic background, including Caucasians, Asians and blacks. Several new alleles were identified containing the coding changes Arg114 His (CYP2C19*9), Pro227 Leu (CYP2C19*10), Arg150 His (CYP2C19*11), stop491 Cys (CYP2C19*12), Arg410 Cys (CYP2C19*13), Leu17 Pro (CYP2C19*14) and Ile19 Leu (CYP2C19*15). When expressed in a bacterial cDNA expression system, CYP2C19*9 exhibited a modest decrease in the V(max) for 4'-hydroxylation of -mephenytoin, and no alteration in its affinity for reductase. CYP2C19*10 exhibited a dramatically higher K(m) and lower V(max) for mephenytoin. CYP2C19*12was unstable and expressed poorly in a bacterial cDNA expression system. Clinical studies will be required to confirm whether this allele is defective in vivo. CYP2C19*9, CYP2C19*10 and CYP2C19*12 all occurred in African-Americans, or individuals of African descent, and represent new potentially defective alleles of CYP2C19 which are predicted to alter risk of these populations to clinically important drugs.