Characterization, isolation, and in vitro culture of leptomeningeal fibroblasts.

Meninges, or the membranous coverings of the brain and spinal cord, play host to dozens of morbid pathologies. In this study we provide a method to isolate the leptomeningeal cell layer, identify leptomeninges in histologic slides, and maintain leptomeningeal fibroblasts in in vitro culture. Using an array of transcriptomic, histological, and cytometric analyses, we identified ICAM1 and SLC38A2 as two novel markers of leptomeningeal cells in vivo and in vitro. Our results confirm the fibroblastoid nature of leptomeningeal cells and their ability to form a sheet-like layer that covers the brain and spine parenchyma. These findings will enable researchers in central nervous system barriers to describe leptomeningeal cell functions in health and disease.

Increased placental nutrient transporter expression at midgestation after maternal growth hormone treatment in pigs: a placental mechanism for increased fetal growth.

Growth hormone (GH) is important in maternal adaptation to pregnancy, and maternal circulating GH concentrations are reduced in human growth-restricted pregnancies. In the pig, maternal GH treatment throughout early to mid pregnancy increases fetal growth, despite constraining effects of adolescent and primiparous pregnancy, high litter size, and restricted maternal nutrition. Because GH cannot cross the placenta and does not increase placental weight, we hypothesized that its effects on fetal growth might be via improved placental structure or function. We therefore investigated effects of maternal GH treatment in pigs on structural correlates of placental function and placental expression of nutrient transporters important to fetal growth. Multiparous (sows) and primiparous pregnant pigs (gilts) were treated with GH (~15 µg kg(-1) day(-1)) or vehicle from Days 25-50 of gestation (n = 7-8 per group, term ~115 days). Placentas were collected at Day 50 of gestation, and we measured structural correlates of function and expression of SLC2A1 (previously known as GLUT1) and SLC38A2 (previously known as SNAT2) nutrient transporters. Maternal GH treatment did not alter placental size or structure, increased protein expression of SLC2A1 in trophoblast (+35%; P = 0.037) and on its basal membrane (+44%; P = 0.011), and increased SLC38A2 protein expression in the basal (+44%; P = 0.001) but not the apical cytoplasm of trophoblast. Our findings suggest that maternal GH treatment increases fetal growth, in part, by enhancing placental nutrient transporter protein expression and hence fetal nutrient supply as well as trophoblast proliferation and differentiation and may have the potential to ameliorate intrauterine growth restriction.

Inner hair cells of mice express the glutamine transporter SAT1.

Glutamate has been implicated in signal transmission between inner hair cells and afferent fibers of the organ of Corti. The inner hair cells are enriched in glutamate and the postsynaptic membranes express AMPA glutamate receptors. However, it is not known whether inner hair cells contain a mechanism for glutamate replenishment. Such a mechanism must be in place to sustain glutamate neurotransmission. Here we provide RT-PCR and immunofluorescence data indicating that system A transporter 1 (SLC38A1), which is associated with neuronal glutamine transport and synthesis of the neurotransmitters GABA and glutamate in CNS, is expressed in inner hair cells. It was previously shown that inner hair cells contain glutaminase that converts glutamine to glutamate. Thus, our finding that inner hair cells express a glutamine transporter and the key glutamine metabolizing enzyme glutaminase, provides a mechanism for glutamate replenishment and bolsters the idea that glutamate serves as a transmitter in the peripheral synapse of the auditory system.

Differential axial localization along the mouse brain vascular tree of luminal sodium-dependent glutamine transporters Snat1 and Snat3.

A specialized brain vasculature is key for establishing and maintaining brain interstitial fluid homeostasis, which for most amino acids (AAs) are ∼10% plasma levels. Indeed, regulation of AA homeostasis seems critical for normal central nervous system functions, and disturbances in brain levels have both direct and indirect roles in several neuropathologies. One mechanism contributing to the plasma to brain AA gradients involves polarized expression of solute carrier (SLC) family transporters on blood-brain barrier (BBB) endothelial cells. Of particular interest is the localization of sodium-dependent transporters that can actively move substrates against their concentration gradient. In this study, the in vivo endothelial membrane localization of the sodium-dependent glutamine transporters Snat3 (Slc38a3) and Snat1 (Slc38a1) was investigated in the mouse brain microvasculature using immunofluorescent colocalization with cellular markers. In addition, luminal membrane expression was probed by in vivo biotinylation. A portion of both Snat3 and Snat1 vascular expressions was localized on luminal membranes. Importantly, Snat1 expression was restricted to larger cortical microvessels, whereas Snat3 was additionally expressed on BBB capillary membranes. This differential expression of system A (Snat1) versus system N (Snat3) transporters suggests distinct roles for Snats in the cerebral vasculature and is consistent with Snat3 involvement in net transendothelial BBB AA transport.

Isolation of plasma membrane vesicles from mouse placenta at term and measurement of system A and system beta amino acid transporter activity.

Placental amino acid transport is essential for optimal fetal growth and development, with a reduced fetal provision of amino acids being implicated as a potential cause of fetal growth restriction (FGR). Understanding placental insufficiency related FGR has been aided by the development of mouse models that have features of the human disease. However, to take maximal advantage of these, methods are required to study placental function in the mouse. Here, we report a method to isolate plasma membrane vesicles from mouse placenta near-term and have used these to investigate two amino acid transporters, systems A and beta, the activities of which are reduced in human placental microvillous plasma membrane (MVM) vesicles from FGR pregnancies. Plasma membrane vesicles were isolated at embryonic day 18 by a protocol involving homogenisation, MgCl(2) precipitation and centrifugation. Vesicles were enriched 11.3+/-0.5-fold in alkaline phosphatase activity as compared to initial homogenate, with minimal intracellular organelle contamination as judged by marker analyses. Cytochemistry revealed alkaline phosphatase was localised between trophoblast layers I and II, with intense reaction product deposited on the maternal-facing plasma membrane of layer II, suggesting that vesicles were derived from this trophoblast membrane. System A and system beta activity in mouse placental vesicles, measured as Na(+)-dependent uptake of (14)C-methylaminoisobutyric acid (MeAIB) and (3)H-taurine respectively confirmed localisation of these transporters to the maternal-facing plasma membrane of layer II. Comparison to human placental MVM showed that system A activity was comparable at initial rate between species whilst system beta activity was significantly lower in mouse. This mirrored the lower expression of TAUT observed in mouse placental vesicles. We conclude that syncytiotrophoblast layer II-derived plasma membrane vesicles can be isolated and used to examine transporter function.

Activation of a system A amino acid transporter, ATA1/SLC38A1, in human hepatocellular carcinoma and preneoplastic liver tissues.

The expression of amino acid transporter (AT) mRNAs including A system (ATA1/SNAT1/SLC38A1, ATA2/SNAT2/SLC38A2 and ATA3/SNAT3/SLC38A4), L system (LAT1/SLC7A5 and LAT2/SLC7A8), and y+ (CAT2/SLC7A2) genes, were compared among hepatocellular carcinoma (HCC) and non-cancerous liver cells. Among them the ATA1 mRNA expression was significantly elevated in all HCC cell lines (HepG2, HLF, HuH7 and JHH4) examined compared with normal liver tissue. We further discovered that the expression of ATA1 mRNA was significantly activated in HCC tissues and also elevated in pre-malignant cirrhotic livers from HCC patients, compared with normal livers from non-HCC patients. The ATA1 protein was extensively accumulated in the cytoplasm of pre-malignant liver and most HCCs, while being weak or undetectably low in normal liver tissues. SiRNA-mediated suppression of endogenous ATA1 lowered the viability of HepG2 cells. Thus, the activation of ATA1 confers growth and survival advantages in pre-malignant and malignant liver lesions.

Expression and adaptive regulation of amino acid transport system A in a placental cell line under amino acid restriction.

Trans-placental transport of amino acids is vital for the developing fetus. Using the BeWo cell line as a placental model, we investigated the effect of restricting amino acid availability on amino acid transport system type A. BeWo cells were cultured either in amino acid-depleted (without non-essential amino acids) or control media for 1, 3, 5 or 6 h. System A function was analysed using alpha(methyl-amino)isobutyric acid (MeAIB) transcellular transport studies. Transporter (sodium coupled neutral amino acid transporter (SNAT1/2)) expression was analysed at mRNA and protein level by Northern and Western blotting respectively. Localisation was carried out using immunocytochemistry. MeAIB transcellular transport was significantly (P < 0.05) increased by incubation of the cells in amino acid-depleted medium for 1 h, and longer incubation times caused further increases in the rate of transfer. However, the initial response was not accompanied by an increase in SNAT2 mRNA; this occurred only after 3 h and further increased for the rest of the 6-h incubation. Similarly, it took several hours for a significant increase in SNAT2 protein expression. In contrast, relocalisation of existing SNAT2 transporters occurred within 30 min of amino acid restriction and continued throughout the 6-h incubation. When the cells were incubated in medium with even lower amino acid levels (without non-essential plus 0.5 x essential amino acids), SNAT2 mRNA levels showed further significant (P < 0.0001) up-regulation. However, incubation of cells in depleted medium for 6 h caused a significant (P = 0.014) decrease in the expression of SNAT1 mRNA. System L type amino acid transporter 2 (LAT2) expression was not changed by amino acid restriction, indicating that the responses seen in the system A transporters were not a general cell response. These data have shown that placental cells adapt in vitro to nutritional stress and have identified the physiological, biochemical and genomic mechanisms involved.

Localization of the sulfate/anion exchanger in the rat liver.

Although the sulfate/anion transporter (sat-1; SLC26A1) was isolated from a rat liver cDNA library by expression cloning, localization of sat-1 within the liver and its contribution to the transport of sulfate and organo sulfates have remained unresolved. In situ hybridization and immunohistochemical studies were undertaken to demonstrate the localization of sat-1 in liver tissue. RT-PCR studies on isolated hepatocytes and liver endothelial and stellate cells in culture were performed to test for the presence of sat-1 in these cells. In sulfate uptake and efflux experiments, the substrate specificity of sat-1 was evaluated. Sat-1 mRNA was found in hepatocytes and endothelial cells. Sat-1 protein was localized in sinusoidal membranes and along the borders of hepatocytes. The canalicular region and bile capillaries were not stained. Sulfate uptake was only slightly affected by sulfamoyl diuretics or organo sulfates. Sulfate efflux from sat-1-expressing oocytes was enhanced in the presence of bicarbonate, indicating sulfate/bicarbonate exchange. Estrone sulfate was not transported by sat-1. Sat-1 may be responsible for the uptake of inorganic sulfate from the blood into hepatocytes to enable sulfation reactions. In hepatocytes and endothelial cells, sat-1 may also supply sulfate for proteoglycan synthesis.

Transforming growth factor-beta 1 stimulates vascular smooth muscle cell L-proline transport by inducing system A amino acid transporter 2 (SAT2) gene expression.

Transforming growth factor-beta1 (TGF-beta 1) is a multifunctional cytokine that contributes to arterial remodelling by stimulating vascular smooth muscle cell (SMC) growth and collagen synthesis at sites of vascular injury. Since l-proline is essential for the synthesis of collagen, we examined whether TGF-beta 1 regulates the transcellular transport of l-proline by vascular SMCs. l-Proline uptake by vascular SMCs was primarily sodium-dependent, pH-sensitive, blocked by neutral amino acids and alpha-(methylamino)isobutyric acid, and exhibited trans-inhibition. Treatment of SMCs with TGF-beta 1 stimulated l-proline transport in a concentration- and time-dependent manner. The TGF-beta 1-mediated l-proline uptake was inhibited by cycloheximide or actinomycin D. Kinetic studies indicated that TGF-beta 1-induced l-proline transport was mediated by an increase in transport capacity independent of any changes in the affinity for l-proline. TGF-beta 1 stimulated the expression of system A amino acid transporter 2 (SAT2) mRNA in a time-dependent fashion that paralleled the increase in l-proline transport. Reverse transcriptase PCR failed to detect the presence of SAT1 or amino acid transporter 3 (ATA3) in either untreated or TGF-beta 1-treated SMCs. These results demonstrate that l-proline transport by vascular SMCs is mediated predominantly by the SAT and that TGF-beta 1 stimulates SMC l-proline uptake by inducing the expression of the SAT2 gene. The ability of TGF-beta 1 to induce SAT2 expression may function to provide SMCs with the necessary levels of l-proline required for collagen synthesis and cell growth.

Biodistribution of [11C] methylaminoisobutyric acid, a tracer for PET studies on system A amino acid transport in vivo.

[N-methyl-11C]alpha-Methylaminoisobutyric acid (11C-MeAIB) is a potentially useful tracer for positron emission tomography (PET) studies on hormonally regulated system A amino acid transport. 11C-MeAIB is a metabolically stable amino acid analogue specific for system A amino acid transport. We evaluated the biodistribution of 11C-MeAIB in rats and humans to estimate the usefulness of the tracer for in vivo human PET studies, for example, on regulation of system A amino acid transport and on tumour imaging. Healthy Sprague-Dawley rats (n=14) were killed 5, 20, 40 or 60 min after the injection of 11C-MeAIB, and the tissue samples were weighed and counted for 11C radioactivity. Ten lymphoma patients with relatively limited tumour burden underwent whole-body (WB) PET imaging with 11C-MeAIB. In addition, three other patients had dynamic PET scanning of the head and neck area, and the tracer uptake was quantitated by calculating the kinetic influx constants (Ki values) for the tracer. In animal studies, the highest activity was detected in the kidney, pancreas, adrenal gland and intestines. In humans, the highest activity was found in the salivary glands, and after that in the kidney and pancreas, similar to the results in animal studies. Rapid uptake was also detected in the skeletal muscle. In the graphical analysis, linear plots were obtained, and the mean fractional tracer uptake values (Ki) of the parotid glands (n=3) and cervical muscles (n=3) were 0.039+/-0.008 min(-1) and 0.013+/-0.006 min(-1), respectively. The Ki value of the tumour (n=1) was 0.064 min(-1). Higher uptake of 11C-MeAIB into the tumour tissue was encountered. These results encourage further 11C-MeAIB PET studies in humans on the physiology and pathology of system A amino acid transport and on tumour detection.