L-system Amino Acid Transporter Research Articles

Early gene activation in chronic leukemic B lymphocytes induced toward a plasma cell phenotype

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of lymphocytes that are arrested at an intermediate stage of B lymphocyte development. CLL B lymphocytes transform (mature) to a plasmacytic phenotype with loss of CD19 and CD20 and the appearance of cytoplasmic immunoglobulin when treated in vitro with phorbol esters. We have used array hybridization technology to describe gene expression patterns for untreated and tetradecanoyl phorbol acetate (TPA)-treated CLL B cells at 5, 10, and 20 min following initial TPA exposure. Three genes, early growth response factor 1 ( EGR-1), dual specificity phosphatase 2, and CD69 (early T-cell activation antigen), showed a 2.0-fold or greater increase in mRNA transcription at four or more of six time points in two studies. Upregulation of expression of these genes was confirmed by real-time polymerase chain reaction in the TPA-treated cells of four CLL patients. A progressive increase in gene expression was observed during the 20-min time course for all three genes. In addition, protein expression of EGR-1 and CD69 was increased as measured by immunofluorescence cell analysis. Several genes (PKC, n-myc, jun D, and BCL-2) previously reported as overexpressed in CLL lymphocytes were overexpressed in these studies also, but were not altered by TPA treatment. Genes for proteins whose upregulation requires hours of TPA exposure (the 4F2hc component of the L-system amino acid transporter, prohibition, and hsp60) were assessed, and their later expression contrasted with the early expression of EGR-1, dual specificity phosphatase 2, and CD69. EGR-1 encodes a zinc-finger transcription factor that is induced by pokeweed mitogen and TPA and promotes B lymphocyte maturation. The dual specificity phosphatase 2 encodes an enzyme that reverses mitogen activated protein kinase cell activation by dephosphorylation. The CD69 protein is induced by TPA in thymocytes and is a type II transmembrane signaling molecule in hematopoietic cells. These findings suggest that the products of these three genes may be central to early steps in the TPA-induced evolution of CLL B cells to a plasmacytic phenotype.

Glial induction of blood-brain barrier-like L-system amino acid transport in the ECV304 cell line.

The blood-brain barrier (BBB) is formed by the presence of tight junction complexes between brain endothelial cells that restrict paracellular permeability. As a consequence, a number of transport proteins are expressed on cerebral endothelial cells to facilitate the transport of nutrients into the brain. Although the modulation of barrier tight junction properties by glial-conditioned medium and by second messengers is well established, little is known about the effects of these factors on carrier-mediated BBB transport processes. The ECV304 cell line shows an endothelial phenotype and can be induced to upregulate certain BBB features in the presence of glial factors. In the present study, we have examined the effect of conditioned medium derived from rat C6-glioma cells (C6CM) on the function of the L-system amino acid transporter in ECV304 cells, using L-leucine as the model substrate, and have determined whether the changes observed can be mimicked by modulating intracellular cAMP levels. ECV304 cells exposed to C6CM exhibited a significant increase in both the affinity of leucine transport and the diffusional constant (Michaelis-Menten), while the maximal transport capacity remained unchanged. Conversely, acute exposure to modulators of the PKA and PKC second messenger pathways was found to reduce significantly the maximal transport capacity and diffusion constants, while transport affinity remained unchanged. In both cases, the maximal flux of leucine was increased, indicating transport of greater efficiency. This study indicates that exposure of ECV304 cells to C6CM provides an influence inducing L-system transport properties characteristic of brain endothelial cells. Furthermore, it appears that L-system-mediated transport of amino acids can be modulated by several distinct pathways.

Evaluation of the RBE4 Cell Line to Explore Carrier-mediated Drug Delivery to the CNS Via the L-system Amino Acid Transporter At the Blood-Brain Barrier

The L-system amino acid transporter on the RBE4 cell line, a well established in vitro model of the blood-brain barrier (BBB), was characterised with the aim to evaluate this in vitro BBB model as tool for the systematic exploration of this endogenous carrier system for drug delivery to the CNS. Transport of l - [3 H]-leucine in RBE4 cells was rapid, Na + -independent, bidirectional and followed the principles of trans -stimulation. The inhibition profile of l -leucine uptake was consistent with transport mediated by the L-system amino acid carrier with strong inhibition by large neutral amino acids (LNAA) such as l -phenylalanine and 2-aminobicyclo-heptanecarboxylic acid (BCH), whereas small neutral, basic and acidic amino acids had no significant effect. The transport of l -leucine into the RBE4 cells was saturable and followed single carrier Michaelis-Menten kinetics with K m 107 ± 10 μ M, V max 9.13 ± 0.45 nmol/min/mg protein and K D 1.36 ± 0.13 μ l/min/mg protein. The kinetic constants of l -leucine transport, as well as the ranking of the kinetic constants of the transport of other LNAA investigated, correspond to those of the BBB in vivo. The characteristics of the LNAA transport in RBE4 cells suggest that transport is mediated by a system with characteristics similar to the L 1 subtype of amino acid transporter, with carrier specificity equivalent to the L 1 carrier system at the BBB in vivo. The study shows that the RBE4 cell line is a very suitable tool for the detailed examination of structure-transport relationships with respect to carrier-mediated drug delivery to the CNS via the L-system amino acid carrier at the BBB. The strength of this in vitro BBB model lies in the combination of the advantages of a cell line, being inexpensive, reproducible and easy to maintain, with the brain endothelium-specific expression of transport systems, to produce an efficient assay for the screening of potential neuropharmaceuticals targeted to specific transport routes to enhance CNS drug delivery.

Gabapentin in the management of convulsive disorders.

Gabapentin, in clinical use since 1993, is indicated as an adjunctive antiepileptic drug (AED) for treatment of complex partial seizures, with or without secondary generalization, in patients over 12 years of age. Although several cellular actions have been described in the literature, the molecular mechanism(s) of action responsible for the anticonvulsant effect of gabapentin has not been conclusively determined. It is likely that gabapentin has multiple concentration-dependent actions that combine in a unique manner to produce antiepileptic efficacy. The pharmacokinetic properties of this water-soluble, amino-acid AED are generally favorable. Absorption appears to be dependent on transport by the L-system amino acid transporter. Elimination of unmetabolized drug occurs by the renal route. Although its therapeutic range is not well characterized, gabapentin has a broad therapeutic index. This implies that a wide range of doses can be used, based on individual patient needs, without significant limitation due to dose-dependent side effects. Gabapentin has few drug-drug interactions, none of which is clinically limiting. Several studies have demonstrated the long-term efficacy of gabapentin with no systematic evidence of tachyphylaxis. In addition, there is increasing evidence to support the use of gabapentin as monotherapy. Gabapentin is safe and is generally well tolerated. To date, nearly 3 million patients have been treated in studies and in open use without causal relationship to a specific life-threatening organ toxicity. Seizure control superior to that observed in well-controlled trials has been reported at higher doses used in clinical practice and in studies. Therefore, gabapentin dosing must be optimized on an individual basis to achieve an adequate trial of the drug and obtain the best seizure control.

Association of HSP60-like Proteins with the L-System Amino Acid Transporter

The carrier protein(s) responsible for mammalian L-system amino acid transport has not been identified. Chronic lymphocytic leukemia (CLL) B-lymphocytes have markedly diminished L-system transport which is restored after treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). Six candidate L-system related plasma membrane proteins were identified in TPA-treated CLL-cells using an L-system photoprobe and ultra-high-resolution two-dimensional gel electrophoresis. In the current studies, two candidate L-system related proteins were obtained from preparative giant two dimensional gels, and the amino acid sequences of peptide fragments from these proteins were determined by Edman degradation. These proteins, forming a doublet on the gels at an apparent size of 40 kDa and pI5.85, had sequence homology to the mitochondrial heat shock protein 60 (hsp60). The presence of these proteins in CLL lymphocyte plasma membranes was confirmed by immunoblotting with antibodies to hsp60. These observations indicate that an hsp60 homologue is associated with the L-system amino acid transporter.

Gabapentin.

The amino acid antiepileptic drug (AED) gabapentin (GBP) is indicated for adjunctive use in the treatment of partial seizures with or without becoming secondarily generalized in individuals older than 12 years. GBP was about as potent as phenytoin in the maximal electroshock test, but had a different profile of efficacy than standard antiepileptics in a range of animal models. Possible mechanisms of action include biochemical effects enhancing the ratio of gamma-aminobutyric acid (GABA) to glutamate, ion-channel actions (direct or indirect), and/ or enhancement of nonsynaptic GABA release. The anticonvulsant effect appears to depend on concentration of gabapentin in neurons, presumably by the L-system amino acid transporter that has been implicated in absorption from the gut. Data from studies for U.S. Food and Drug Administration (FDA) approval suggested a direct relationship of clinical response to dose and efficacy did not plateau at the doses used. The maximally effective dose, relationship of efficacy to blood level, and maximum tolerable dose are not yet known conclusively. Lack of significant binding to plasma proteins and lack of liver metabolism contribute to the absence of known limiting drug-drug interactions, particularly with other AEDs. Excretion intact in the urine affords dose adjustment on the basis of creatinine clearance. A half-life of approximately 7 h necessitates multiple doses daily for many individuals. The medication is well tolerated, in general. Side effects tend to be mild to moderate in intensity, most frequently affect the central nervous system, and resolve with time in many individuals. GBP has been prescribed for approximately 70,000 individuals worldwide without untoward incidence of severe systemic toxicity to date. Safety data continue to accumulate. GBP has been labeled category C on the basis of effects on rodent fetuses. Experience with use in pregnant women is limited and human teratogenic effects have not been reported. Data from ongoing monotherapy trials will help to clarify the range of clinical utility of gabapentin.

Phorbol ester-induced membrane proteins in chronic leukemic B-lymphocytes. Candidate proteins for the L-system amino acid transporter

Chronic lymphocytic leukemia (CLL) B-lymphocytes have markedly diminished membrane L-system amino acid transport as compared with normal mature B- and T-lymphocytes. L-system functional recovery is induced in CLL B-cells by the maturational agent, 12-O-tetradecanoylphorbol-13-acetate (TPA). The studies reported here extend the analysis of CLL B-cell maturation by comparing membrane protein expression in untreated and TPA-treated CLL B-cells, with the identification of candidate proteins for the L-system transporter. Cell membrane proteins of resting and TPA-treated CLL B-lymphocytes were studied using ultrahigh resolution giant two-dimensional gel electrophoresis. Cellular proteins were metabolically labeled with [35S]methionine, and, in separate experiments, membrane proteins were photoaffinity labeled with [125I] iodoazidophenylalanine, an amino acid transporter by the L-system and which binds at or near the L-system transport carrier. In a partially purified membrane preparation, approximately 1400 proteins were identified by metabolic labeling. Following TPA treatment for 17 h, 14 new metabolically labeled membrane proteins were identified, and five of these also were labeled by the L-system photoprobe. Photolabeling of four of these proteins was inhibited by an excess of the L-system prototype amino acid, 2-aminobicyclo(2.2.1)heptane-2-carboxylic acid. Given these labeling characteristics, one or more of these four proteins may be related to the L-system amino acid transport carrier.

Photoinhibition of 2-Amino-2-carboxybicyclo[2,2,1]heptane Transport by O-Diazoacetyl-L-serine: An Initial Step in Identifying the L-system Amino Acid Transporter

Neutral amino acid uptake into mammalian cells occurs predominantly through the L, A, and ASC carrier-mediated transport systems. The proteins responsible for transport by these systems have not been isolated, and the three pathways presently are defined by their amino acid specificity and physiologic parameters. We have found that the amino acid derivative, O-diazoacetyl-L-serine (azaserine), is a potentially useful probe for identification of the L-(leucine-favoring) system transporter in human T-lymphocytes. Uptake of azaserine competitively inhibits the uptake of the prototype L-system amino acid, 2-amino-2-carboxybicycloheptane (BCH). Azaserine undergoes photolytic cleavage with 365 nm incident light to yield a highly reactive carbene intermediate and free N2. Following photolysis of [14C]azaserine in a suspension of lymphocytes, the 14C label is detectable within a crude cytoplasmic membrane preparation, and this process is inhibited by a 50-fold excess of unlabeled azaserine or 2-amino-2-carboxybicycloheptane, suggesting that the 14C-product is associated with the membranes at or near the L-system transport site. Furthermore, photolysis of azaserine in the presence of lymphocytes results in specific irreversible inhibition of L-system transport. Thus, photolysis of azaserine provides an initial step toward the identification of the L-system transporter.

Y+- and L-system amino acid transport in normal and chronic lymphocytic leukemia lymphocytes: Photoinhibition by fluoronitrophenylazide

Three major pathways mediate amino acid transport into mammalian cells: the Asystem and the ASC-system, which require a sodium gradient across the plasma membrane, and the L-system, which has no requirement for a sodium gradient. We have found that the lymphocytes from patients with B-cell chronic lymphocytic leukemia (CLL) have a marked reduction in the L-system of amino acid transport when compared to normal human B-lymphocytes from blood or tonsils. Transport by the A- and ASCsystems was not decreased in CLL B-lymphocytes. Because of the specific defect of the sodium-independent L-system amino acid transport in CLL cells, we have examined the activity of another sodium-independent transport system, the Y+-system, in human lymphocytes. The Y+-system favors the transport of dibasic, cationic amino acids such as lysine, ornithine, and arginine, which carry a positively charged group on their side chains. Our studies indicate that there is a large nonsaturable component of amino acid transport by the Y+-system in human lymphocytes. Using a multicomponent mathematical analysis, we have determined that the saturable component of Y+-transport is similar in T- (thymus-derived) and B- (bone-marrow-derived) lymphocytes and is unimpaired in CLL B-lymphocytes. Further, fluoronitrophenylazide, which was thought to be a specific inhibitor of the Y+-system when photoactivated, also inhibits A-, and L-system transport in CLL, T-, and B-lymphocytes.

A multicomponent analysis of amino acid transport systems in human lymphocytes. 1. Kinetic parameters of the A and L systems and pathways of uptake of naturally occurring amino acids in blood lymphocytes.

We have determined the kinetic parameters of natural and system-specific synthetic amino acid transport by human blood lymphocytes, using a multi-component computer analysis that separates carrier-mediated uptake from diffusion. These studies were initiated in order to provide the basis for studies of human blood T and B lymphocytes and malignant lymphocytes. Methylaminoisobutyric acid (methyl-AIB) and 2-amino-2-carboxy-bicyclo (2,2,1) heptane (BCH) uptakes into lymphocytes were measured as prototypes of A- and L-system amino acid transport. The Michaelis constant for methyl-AIB uptake was 540 microM; the maximal velocity of uptake was 28 mumol/L cell water/min, and the diffusion coefficient was .004 min-1. In contrast, the Michaelis constant for BCH uptake was 63 microM; the maximal velocity was 969 mumol/L cell water/min, and the diffusion coefficient was .141 min-1. The transport of the naturally occurring amino acids, alanine, proline, and leucine was defined by studies of: (1) competitive inhibition with the system-specific synthetic amino acids, methyl-AIB and BCH, (2) the effect of the transcellular sodium gradient on transport, and (3) evaluation of the time-dependent increase of transport in amino acid-deficient medium (adaptation). Alanine was transported principally (approximately 70%) by the ASC-system, and leucine was transported principally (70%) by the L-system in lymphocytes. The analysis of proline transport was more complex because of a large component of uptake by diffusion even at low amino acid concentrations. Taken together, the kinetics of sodium-sensitive uptake and the results of competitive inhibition studies indicated that proline was transported by the A-system (30%), the ASC system (30%), and also by the L-system (15%).

Amino acid transport in bone: stimulation by cyclic AMP.

ARTICLESAmino acid transport in bone: stimulation by cyclic AMPJM Phang, and SJ DowningJM Phang, and SJ DowningPublished Online:01 Jan 1973https://doi.org/10.1152/ajplegacy.1973.224.1.191MoreSectionsPDF (1 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByParathyroid hormone (PTH) regulation of metabolic homeostasis: An old dog teaches us new tricksMolecular Metabolism, Vol. 60SLC1A5 provides glutamine and asparagine necessary for bone development in mice14 October 2021 | eLife, Vol. 10The role of osteoblasts in energy homeostasis28 August 2019 | Nature Reviews Endocrinology, Vol. 15, No. 11Wnt signaling and cellular metabolism in osteoblasts26 November 2016 | Cellular and Molecular Life Sciences, Vol. 74, No. 9Effect of theophylline on l-alanine absorption in the rat jejunumFood and Chemical Toxicology, Vol. 42, No. 12Glial induction of blood-brain barrier-Like L-system amino acid transport in the ECV304 cell line6 June 2002 | Glia, Vol. 39, No. 2The regulation of L-proline transport by insulin-like growth factor-I in human osteoblast-like SaOS-2 cellsPflügers Archiv - European Journal of Physiology, Vol. 432, No. 3Prenatal effects of maternal caffeine intake and dietary high protein on mandibular development in fetal rats9 March 2007 | British Journal of Nutrition, Vol. 63, No. 2Parathyroid hormone alteration of free and tRNA-bound proline specific activities in cultured mouse osteoblast-like cellsBiochemical and Biophysical Research Communications, Vol. 161, No. 3Parathyroid hormone regulation of proline uptake by cultured neonatal mouse osteoblastlike cells3 December 2009 | Journal of Bone and Mineral Research, Vol. 4, No. 1Effect of parathyroid hormone on amino acid transport by cultured neonatal mouse calvarial bone cells3 December 2009 | Journal of Bone and Mineral Research, Vol. 3, No. 2Characteristics of a neutral amino acid transport system (system A) in osteoblastic rat osteosarcoma cellsExperimental Cell Research, Vol. 169, No. 2The effect of prostaglandin E2 (PGE2) on amino acid uptake and protein formation by lung fibroblasts.Journal of Biological Chemistry, Vol. 261, No. 19Effects of maternal caffeine intake on the growth of rat tooth germs in protein-energy malnourished neonatesArchives of Oral Biology, Vol. 30, No. 2Effects of amino acids, glucagon, insulin and acetylcholine on cyclic nucleotide metabolism and amylase secretion in isolated mouse pancreatic fragmentsBiochemical Pharmacology, Vol. 32, No. 13The regulation of neutral amino acid transport in mammalian cellsBiochimica et Biophysica Acta (BBA) - Reviews on Biomembranes, Vol. 737, No. 2Effects of Insulin on Intracellular FunctionsRegulation of chondrosarcoma metabolism by cyclic adenosine 3'5'-monophosphateW. D. McCumbee, and H. E. Lebovitz1 September 1980 | American Journal of Physiology-Endocrinology and Metabolism, Vol. 239, No. 3Effects of dibutyryl cyclic amp on the transport of α-methyl-d-glucoside and α-aminoisobutyric acid in separated tubules and brush border membranes from rabbit kidneyBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 558, No. 1The regulation of amino acid transport in animal cellsBiochimica et Biophysica Acta (BBA) - Reviews on Biomembranes, Vol. 515, No. 4Receptor mediated glucocorticoid inhibition of protein synthesis in isolated bone cellsJournal of Steroid Biochemistry, Vol. 9, No. 3HypoparathyroidismHormonal Regulation of Bone FormationThe actions of parathyroid hormone on bone: Relation to bone remodeling and turnover, calcium homeostasis, and metabolic bone diseaseMetabolism, Vol. 25, No. 9Effects of Na+ depletion, theophylline and 2,4-dinitrophenol on neutral amino acid influx and cyclic adenosine 3′:5′-monophosphate levels in chicken small intestineComparative Biochemistry and Physiology Part C: Comparative Pharmacology, Vol. 53, No. 2Stimulation of cartilage amino acid uptake by growth hormone-dependent factors in serumBiochimica et Biophysica Acta (BBA) - General Subjects, Vol. 381, No. 2Transport of Amino Acids in Brain SlicesBiochemistry of Bone DiseasesEffect of theophylline and Na+ on methionine influx in Na+-depleted intestineBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 373, No. 2Parathyroid hormone and calcitonin regulation of renal functionKidney International, Vol. 6, No. 5 More from this issue > Volume 224Issue 1January 1973Pages 191-196 Copyright & PermissionsCopyright © 1973 by American Physiological Societyhttps://doi.org/10.1152/ajplegacy.1973.224.1.191PubMed4345737History Published online 1 January 1973 Published in print 1 January 1973 Metrics