Intracellular uptake and inhibitory activity of aromatic fluorinated amino acids in human breast cancer cells.

Nonproteinogenic amino acids that either occur naturally or are synthesized chemically are becoming important tools in modern drug discovery. In this context, fluorinated amino acids have great potential in the development of novel pharmaceuticals and drugs. To assess whether different fluorinated aromatic amino acid analogues of phenylalanine, tyrosine, and tryptophan are potentially interesting as therapeutic drugs, we examined their cytostatic and cytotoxic effects on the growth of the human breast cancer cell line MCF-7. Of all the tested analogues L-4-fluorotryptophan, L-6-fluorotryptophan and L-p-fluorophenylalanine effectively and irreversibly inhibited cell growth with IC(50) values in the low micromolar range (3-15 microM). Additionally, using L-4-[14C]fluorotryptophan, and L-6-[14C]fluorotryptophan, we discovered that the cellular uptake of these fluorinated amino acids occurs through active transport with a 70-fold excess of intracellular over extracellular concentrations. We identified system L as the responsible amino acid transporter. Our findings fully support the idea that fluorinated aromatic amino acid analogues are promising chemotherapeutics with the potential for use in combination with classical cancer therapy, and as new cytotoxic drugs for certain tumor types such as melanoma.

Functional activity of a large neutral amino acid transporter (LAT) in rabbit retina: a study involving the in vivo retinal uptake and vitreal pharmacokinetics of L-phenyl alanine.

PURPOSE: The purpose of this study is to elucidate the functional activity of large neutral amino acid transporter (LAT) in rabbit retina and to delineate its role in the retinal uptake and intravitreal pharmacokinetics of L-phenylalanine (L-Phe). METHODS: In vivo retinal uptake of L-Phe and L-alanine (L-Ala) was determined in the presence and absence of specific transport inhibitors following intravitreal administration. L and D isomers of amino acids were employed as inhibitors to determine the stereo-selectivity of LAT. Reverse transcription-polymerase chain reaction (RT-PCR) was carried out for LAT isoforms (LAT1 and LAT2). Vitreal disposition of L-Phe following administration in rabbit vitreous was studied in the presence of an other competing LAT substrate D-methionine using microdialysis. RESULTS: Retinal uptake of L-Phe was significantly inhibited in presence of a specific LAT inhibitor, 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid (BCH), L isomers of large neutral amino acids and LAT1 specific but not by LAT2 specific and charged amino acids. No significant inhibition of L-Ala retinal uptake was observed with LAT substrates. LAT isoforms (LAT1 and LAT2) were identified by RT-PCR in rabbit retina. The mean residence time (MRT) and area under curve (AUC) values of L-Phe following intravitreal administration were significantly increased in the presence of D-methionine, a LAT substrate. CONCLUSIONS: This study demonstrates the functional activity and molecular expression of large neutral amino acid transporter in the rabbit retina. Furthermore, based on these studies it can be concluded that LAT is involved in the retinal uptake and intravitreal elimination of L-Phe.

Mammalian target of rapamycin in the human placenta regulates leucine transport and is down-regulated in restricted fetal growth.

Pathological fetal growth is associated with perinatal morbidity and the development of diabetes and cardiovascular disease later in life. Placental nutrient transport is a primary determinant of fetal growth. In human intrauterine growth restriction (IUGR) the activity of key placental amino acid transporters, such as systems A and L, is decreased. However the mechanisms regulating placental nutrient transporters are poorly understood. We tested the hypothesis that the mammalian target of rapamycin (mTOR) signalling pathway regulates amino acid transport in the human placenta and that the activity of the placental mTOR pathway is reduced in IUGR. Using immunohistochemistry and culture of trophoblast cells, we show for the first time that the mTOR protein is expressed in the transporting epithelium of the human placenta. We further demonstrate that placental mTOR regulates activity of the l-amino acid transporter, but not system A or taurine transporters, by determining the mediated uptake of isotope-labelled leucine, methylaminoisobutyric acid and taurine in primary villous fragments after inhibition of mTOR using rapamycin. The protein expression of placental phospho-S6K1 (Thr-389), a measure of the activity of the mTOR signalling pathway, was markedly reduced in placentas obtained from pregnancies complicated by IUGR. These data identify mTOR as an important regulator of placental amino acid transport, and provide a mechanism for the changes in placental leucine transport in IUGR previously demonstrated in humans. We propose that mTOR functions as a placental nutrient sensor, matching fetal growth with maternal nutrient availability by regulating placental nutrient transport.

Down-regulation of N-type voltage-activated Ca2+ channels by gabapentin.

1. Although the cellular and molecular mechanisms of the anticonvulsant action of gabapentin (GBP) remain incompletely described, in vitro studies have shown that GBP binds to the alpha2delta subunit of the high voltage-activated (HVA) Ca2+ channels. 2. In this report, we analyzed the effects of GBP on the functional expression of HVA Ca2+ channels in the PC12 cell line model system. Negligible inhibition of Ca2+ channel activity was observed after acute treatment, but a significant decrease in Ca2+ current amplitude was promoted by chronic exposure to GBP. 3. Consistent with this, radioligand binding experiments showed a comparable reduction in the total number of membrane HVA N-type channels after GBP treatment.

S-adenosylmethionine is substrate for carrier mediated transport at the blood-brain barrier in vitro.

S-adenosylmethionine (SAM) is the sole methyl donor in the CNS where it is involved in a multitude of biochemical reactions. Peripherally administered SAM has been shown to increase SAM levels in cerebrospinal fluid and is reported to be effective in the treatment of numerous neurological disorders suggesting SAM crosses the blood-brain barrier (BBB). The mechanism of SAM entry into the brain remains unknown, but the presence of adenosyl and methionine residues in the molecule suggests probable entry via carrier mediated transport. We have investigated whether SAM utilises endogenous transport systems in cerebral endothelial cells, using RBE4 cells, an in vitro model of the BBB. SAM did not influence the transport of [(3)H]-methionine and only marginally reduced the uptake of [(3)H]-leucine in RBE4 cells. The inhibition constant for the latter was 2.11+/-0.29 mM (mean+/-S.E.M.). However, increasing concentrations of SAM strongly inhibited the transport of [3H]-adenosine in RBE4 cells in both the presence and the absence of sodium in the medium, with K(i) values of 199+/-32 and 139+/-8.4 microM, respectively. Lineweaver-Burk plots suggest a competitive mode of inhibition. The findings suggest that SAM is not recognised by the L-system transporter for large neutral amino acids at the brain endothelium. A significant interaction with the transport of adenosine, however, indicates that SAM has affinity for the nucleoside carrier systems; this is within the range of K(m) values of natural substrates and suggest that SAM may enter the CNS via the Na(+)-independent nucleoside carrier systems at the brain capillary endothelium.