Differential influence of cAMP on the expression of the three subtypes (ATA1, ATA2, and ATA3) of the amino acid transport system A

AbstractTreatment of HepG2 cells with forskolin led to 60–100% stimulation of system A activity, measured as the Na+-dependent uptake of α-(methylamino)isobutyric acid. The stimulation was reproducible with cholera toxin and dibutyryl cAMP, and inhibitable by H7, a non-specific protein kinase inhibitor. The stimulatory effect was eliminated by cycloheximide and actinomycin D. The forskolin effect was associated with an increase in the maximal velocity of the transport system, with no change in substrate affinity. These cells express three different subtypes of system A (ATA1, ATA2, and ATA3). Treatment with forskolin increased the steady-state levels of ATA1 and ATA2 mRNAs, but decreased that of ATA3 mRNA

Structure and function of ATA3, a new subtype of amino acid transport system A, primarily expressed in the liver and skeletal muscle

AbstractTo date, two different transporters that are capable of transporting α-(methylamino)isobutyric acid, the specific substrate for amino acid transport system A, have been cloned. These two transporters are known as ATA1 and ATA2. We have cloned a third transporter that is able to transport the system A-specific substrate. This new transporter, cloned from rat skeletal muscle and designated rATA3, consists of 547 amino acids and has a high degree of homology to rat ATA1 (47% identity) and rat ATA2 (57% identity). rATA3 mRNA is present only in the liver and skeletal muscle. When expressed in Xenopus laevis oocytes, rATA3 mediates the transport of α-[14C](methylamino)isobutyric acid and [3H]alanine. With the two-microelectrode voltage clamp technique, we have shown that exposure of rATA3-expressing oocytes to neutral, short-chain aliphatic amino acids induces inward currents. The amino acid-induced current is Na+-dependent and pH-dependent. Analysis of the currents with alanine as the substrate has shown that the K0.5 for alanine (i.e., concentration of the amino acid yielding half-maximal current) is 4.2±0.1 mM and that the Na+:alanine stoichiometry is 1:1

Transport of N-acetylaspartate via murine sodium/dicarboxylate cotransporter NaDC3 and expression of this transporter and aspartoacylase II in ocular tissues in mouse

AbstractCanavan disease is a genetic disorder associated with optic neuropathy and the metabolism of N-acetylaspartate is defective in this disorder due to mutations in the gene coding for the enzyme aspartoacylase II. Here we show that the plasma membrane transporter NaDC3, a Na+-coupled transporter for dicarboxylates, is able to transport N-acetylaspartate, suggesting that the transporter may function in concert with aspartoacylase II in the metabolism of N-acetylaspartate. Since Canavan disease is associated with ocular complications, we investigated the expression pattern of NaDC3 and aspartoacylase II in ocular tissues in mouse by in situ hybridization. These studies show that NaDC3 mRNA is expressed in the optic nerve, most layers of the retina, retinal pigment epithelium, ciliary body, iris, and lens. Aspartoacylase II mRNA is coexpressed in most of these cell types. We conclude that transport of N-acetylaspartate into ocular tissues via NaDC3 and its subsequent hydrolysis by aspartoacylase II play an essential role in the maintenance of visual function

Reduced-folate carrier (RFC) is expressed in placenta and yolk sac, as well as in cells of the developing forebrain, hindbrain, neural tube, craniofacial region, eye, limb buds and heart

BACKGROUND: Folate is essential for cellular proliferation and tissue regeneration. As mammalian cells cannot synthesize folates de novo, tightly regulated cellular uptake processes have evolved to sustain sufficient levels of intracellular tetrahydrofolate cofactors to support biosynthesis of purines, pyrimidines, and some amino acids (serine, methionine). Though reduced-folate carrier (RFC) is one of the major proteins mediating folate transport, knowledge of the developmental expression of RFC is lacking. We utilized in situ hybridization and immunolocalization to determine the developmental distribution of RFC message and protein, respectively. RESULTS: In the mouse, RFC transcripts and protein are expressed in the E10.0 placenta and yolk sac. In the E9.0 to E11.5 mouse embryo RFC is widely detectable, with intense signal localized to cell populations in the neural tube, craniofacial region, limb buds and heart. During early development, RFC is expressed throughout the eye, but by E12.5, RFC protein becomes localized to the retinal pigment epithelium (RPE). CONCLUSIONS: Clinical studies show a statistical decrease in the number of neural tube defects, craniofacial abnormalities, cardiovascular defects and limb abnormalities detected in offspring of female patients given supplementary folate during pregnancy. The mechanism, however, by which folate supplementation ameliorates the occurrence of developmental defects is unclear. The present work demonstrates that RFC is present in placenta and yolk sac and provides the first evidence that it is expressed in the neural tube, craniofacial region, limb buds and heart during organogenesis. These findings suggest that rapidly dividing cells in the developing neural tube, craniofacial region, limb buds and heart may be particularly susceptible to folate deficiency

Retraction Note: Identification of a novel Na+-coupled Fe3+-citrate transport system, distinct from mammalian INDY, for uptake of citrate in mammalian cells.

This paper has been retracted at the request of the authors

Cloning and functional characterization of a Na+-independent, broad-specific neutral amino acid transporter from mammalian intestine

AbstractWe have isolated a cDNA from a rabbit intestinal cDNA library which, when co-expressed with the heavy chain of the human 4F2 antigen (4F2hc) in mammalian cells, induces system L-like amino acid transport activity. This protein, called LAT2, consists of 535 amino acids and is distinct from LAT1 which also interacts with 4F2hc to induce system L-like amino acid transport activity. LAT2 does not interact with rBAT, a protein with a significant structural similarity to 4F2hc. The 4F2hc/LAT2-mediated transport process differs from the 4F2hc/LAT1-mediated transport in substrate specificity, substrate affinity, tissue distribution, interaction with D-amino acids, and pH-dependence. The 4F2hc/LAT2-associated transport process has a broad specificity towards neutral amino acids with Kt values in the range of 100–1000 μM, does not interact with D-amino acids to any significant extent, and is stimulated by acidic pH. In contrast, the 4F2hc/LAT1-associated transport process has a narrower specificity towards neutral amino acids, but with comparatively higher affinity (Kt values in the range of 10–20 μM), interacts with some D-amino acids with high affinity, and is not influenced by pH. LAT2 is expressed primarily in the small intestine and kidney, whereas LAT1 exhibits a much broader tissue distribution

Hydroxyurea differentially modulates activator and repressors of γ-globin gene in erythroblasts of responsive and non-responsive patients with sickle cell disease in correlation with Index of Hydroxyurea Responsiveness

Hydroxyurea (HU), the first of two drugs approved by the US Food and Drug Administration for treating patients with sickle cell disease (SCD), produces anti-sickling effect by re-activating fetal γ-globin gene to enhance production of fetal hemoglobin. However, approximately 30% of the patients do not respond to HU therapy. The molecular basis of non-responsiveness to HU is not clearly understood. To address this question, we examined HU-induced changes in the RNA and protein levels of transcription factors NF-Y, GATA-1, -2, BCL11A, TR4, MYB and NF-E4 that assemble the γ-globin promoter complex and regulate transcription of γ-globin gene. In erythroblasts cultured from peripheral blood CD34+ cells of patients with SCD, we found that HU-induced changes in the protein but not the RNA levels of activator GATA-2 and repressors GATA-1, BCL11A and TR4 correlated with HU-induced changes in fetal hemoglobin (HbF) levels in the peripheral blood of HU high and low responders. However, HU did not significantly induce changes in the protein or RNA levels of activators NF-Y and NF-E4. Based on HU-induced changes in the protein levels of GATA-2, -1 and BCL11A, we calculated an Index of Hydroxyurea Responsiveness (IndexHU-3). Compared to the HU-induced fold changes in the individual transcription factor protein levels, the numerical values of IndexHU-3 statistically correlated best with the HU-induced peripheral blood HbF levels of the patients. Thus, IndexHU-3 can serve as an appropriate indicator for inherent HU responsiveness of patients with SCD

Transport of the Photodynamic Therapy Agent 5-Aminolevulinic Acid by Distinct H ϩ -Coupled Nutrient Carriers Coexpressed in the Small Intestine

ABSTRACT 5-Aminolevulinic acid (ALA) is a prodrug used in photodynamic therapy, fluorescent diagnosis, and fluorescent-guided resection because it leads to accumulation of the photosensitizer protoporphyrin IX (PpIX) in tumor tissues. ALA has good oral bioavailability, but high oral doses are required to obtain selective PpIX accumulation in colonic tumors because accumulation is also observed in normal gut mucosa. Structural similarities between ALA and GABA led us to test the hypothesis that the H ϩ -coupled amino acid transporter PAT1 (SLC36A1) will contribute to luminal ALA uptake. Radiolabel uptake and electrophysiological measurements identified PAT1-mediated H ϩ -coupled ALA symport after heterologous expression in Xenopus oocytes. The selectivity of the nontransported inhibitors 5-hydroxytryptophan and 4-aminomethylbenzoic acid for, respectively, PAT1 and the H ϩ -coupled di/tripeptide transporter PepT1 (SLC15A1) were examined. 5-Hydroxytryptophan selectively inhibited PAT1-mediated amino acid uptake across the brush-border membrane of the human intestinal (Caco-2) epithelium whereas 4-aminomethylbenzoic acid selectively inhibited PepT1-mediated dipeptide uptake. The inhibitory effects of 5-hydroxytryptophan and 4-aminomethylbenzoic acid were additive, demonstrating that both PAT1 and PepT1 contribute to intestinal transport of ALA. This is the first demonstration of overlap in substrate specificity between these distinct transporters for amino acids and dipeptides. PAT1 and PepT1 expression was monitored by reverse transcriptase-polymerase chain reaction using paired samples of normal and cancer tissue from human colon. mRNA for both transporters was detected. PepT1 mRNA was increased 2.3-fold in cancer tissues. Thus, increased PepT1 expression in colonic cancer could contribute to the increased PpIX accumulation observed. Selective inhibition of PAT1 could enhance PpIX loading in tumor tissue relative to that in normal tissue