Cortisol stimulates system A amino acid transport and SNAT2 expression in a human placental cell line (BeWo)

2006 ◽  
Vol 291 (3) ◽  
pp. E596-E603 ◽  
Author(s):  
Helen N. Jones ◽  
Cheryl J. Ashworth ◽  
Ken R. Page ◽  
Harry J. McArdle
Keyword(s):  
Amino Acid ◽  
Cell Line ◽  
Expression System ◽  
Isobutyric Acid ◽  
Fetal Plasma ◽  
Functional Studies ◽  
Protein Levels ◽  
System A ◽  
Cortisol Levels ◽  
Amino Isobutyric Acid

Both placental system A activity and fetal plasma cortisol concentrations are associated with intrauterine growth retardation, but it is not known if these factors are mechanistically related. Previous functional studies using hepatoma cells and fibroblasts produced conflicting results regarding the regulation of system A by cortisol. Using the b30 BeWo choriocarcinoma cell line, we investigated the regulation of system A by cortisol. System A function was analyzed using methyl amino isobutyric acid (MeAIB) transcellular transport studies. Transporter expression [system A transporter (SNAT)1/2] was studied at the mRNA and protein levels using Northern and Western blotting, respectively. Localization was carried out using immunocytochemistry. The [14C]MeAIB transfer rate across BeWo monolayers after preincubation with cortisol for 24 h was significantly increased compared with control. This was associated with a relocalization of the SNAT2 transporter at lower cortisol levels and significant upregulation of mRNA and protein expression levels at cortisol levels >1 μM. This is the first study to show functional and molecular regulation of system A by cortisol in BeWo cells. It is also the first study to identify which system A isoform is regulated. These results suggest that cortisol may be involved in upregulation of system A in the placenta to ensure sufficient amino acid supply to the developing fetus.

scholarly journals Insulin-induced formation of ruffling membranes of KB cells and its correlation with enhancement of amino acid transport.

1984 ◽  
Vol 98 (3) ◽  
pp. 801-809 ◽  
Author(s):  
K Goshima ◽  
A Masuda ◽  
K Owaribe
Keyword(s):  
Amino Acid ◽  
Cyclic Amp ◽  
Close Correlation ◽  
Isobutyric Acid ◽  
Dibutyryl Cyclic Amp ◽  
Kb Cells ◽  
System A ◽  
A Cell ◽  
Amino Isobutyric Acid ◽  
Human Epidermoid Carcinoma

Insulin induced the formation of ruffling membranes in cultured KB cells (a cell strain derived from human epidermoid carcinoma) within 1-2 min after its addition. The ruffled regions were stained strongly with antibody to actin but not that to tubulin. Pretreatment of KB cells with agents disrupting microfilaments (cytochalasins), but not with those disrupting microtubules (colcemid, nocodazole, and colchicine) completely inhibited the formation of ruffling membranes. Pretreatment of KB cells with dibutyryl cyclic AMP, but not with dibutyryl cyclic GMP, also inhibited the formation of ruffling membranes. Addition of insulin enhanced Na+-dependent uptake of a system A amino acid (alpha-amino isobutyric acid; AIB) by the cells within 5 min after the addition, and decreased the cyclic AMP content of the cells. Treatments that inhibited insulin-induced formation of ruffling membranes of KB cells also inhibited insulin-induced enhancement of their AIB uptake. From these observations, the mechanism of insulin-induced formation of ruffling membranes and its close correlation with AIB transport are discussed.

Somatostatin inhibits insulin-stimulated amino acid uptake into cultured rat myoblasts

1987 ◽  
Vol 114 (4) ◽  
pp. 470-474 ◽  
Author(s):  
G. S. G. Spencer ◽  
D. J. Hill ◽  
G. J. Garssen ◽  
J. P. G. Williams
Keyword(s):  
Amino Acid ◽  
Nutrient Uptake ◽  
Monolayer Culture ◽  
Amino Acid Uptake ◽  
Inhibitory Effect ◽  
Isobutyric Acid ◽  
Acid Uptake ◽  
Newborn Rat ◽  
Amino Isobutyric Acid

Abstract. The effects of somatostatin on the acute metabolic actions of insulin on newborn rat myoblasts in culture has been examined during monolayer culture. Somatostatin significantly inhibited the insulin-stimulated uptake of [3H]leucine and [3H]amino-isobutyric acid into myoblasts but had no effect on basal (unstimulated) uptake of these two substances. The lowest concentration of somatostatin to have a significant effect was 10 μg/l, and this was apparent in all the experiments undertaken. The inhibitory effect of somatostatin was seen at all effective concentrations of insulin used (0.3–1 U/l). These findings lend support to the concept of an endocrine role for somatostatin in vivo and suggest that a peripheral antagonism may exist between circulating insulin and somatostatin on anabolic processes such as nutrient uptake into cells.

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

Reproduction ◽  
2006 ◽  
Vol 131 (5) ◽  
pp. 951-960 ◽  
Author(s):  
H N Jones ◽  
C J Ashworth ◽  
K R Page ◽  
H J McArdle
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cell Line ◽  
Transport System ◽  
Amino Acid Transport ◽  
Essential Amino Acids ◽  
Acid Transport ◽  
Amino Acid Transport System ◽  
Transcellular Transport ◽  
System A

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 α(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 × 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 adaptin vitroto nutritional stress and have identified the physiological, biochemical and genomic mechanisms involved.

Angiotensin II decreases system A amino acid transporter activity in human placental villous fragments through AT1 receptor activation

2006 ◽  
Vol 291 (5) ◽  
pp. E1009-E1016 ◽  
Author(s):  
Eiji Shibata ◽  
Robert W. Powers ◽  
Augustine Rajakumar ◽  
Frauke von Versen-Höynck ◽  
Marcia J. Gallaher ◽  
...  
Keyword(s):  
Amino Acid ◽  
Angiotensin Ii ◽  
Atpase Activity ◽  
Renin Angiotensin System ◽  
Coupled System ◽  
Receptor Activation ◽  
Isobutyric Acid ◽  
Transport Systems ◽  
System A ◽  
Placental Villi

Reduced transport of amino acids from mother to fetus can lead to fetal intrauterine growth restriction (IUGR). The activities of several amino acid transport systems, including system A, are decreased in placental syncytiotrophoblast of IUGR pregnancies. Na+-K+-ATPase activity provides an essential driving force for Na+-coupled system A transport, is decreased in the placenta of IUGR pregnancies, and is decreased by angiotensin II in several tissues. Several reports have shown activation of the fetoplacental renin-angiotensin system (RAS) in IUGR. We investigated the effect of angiotensin II on placental system A transport and Na+-K+-ATPase activity in placental villi. Placental system A activity in single primary villous fragments was measured as the Na+-dependent uptake of α-(methylamino)isobutyric acid, and Na+/K+ ATPase activity was measured as ouabain-sensitive uptake of 86rubidium. Angiotensin II decreased system A activity in a concentration-dependent fashion (10–500 nmol/l). Angiotensin II type 1 receptor (AT1-R) antagonists losartan and AT1-R anti-peptide blocked the angiotensin II effect, but the angiotensin II type 2 receptor antagonist PD-123319 was without effect. System A activity was not altered by preincubation with AT1-R-independent vasoconstrictors, and antioxidants did not prevent the decrease in activity mediated by angiotensin II. Angiotensin II decreased Na+-K+-ATPase activity by an AT1-R dependent mechanism, and inhibition of Na+-K+-ATPase activity decreased system A activity in a dose-response fashion. These data suggest that angiotensin II, via AT1-R signaling, decreases system A activity by suppressing Na+-K+-ATPase in human placental villi, consistent with possible adverse effects of enhanced placental RAS on fetal growth.

Effects of phenylarsine oxide on insulin-stimulated system A amino acid uptake in skeletal muscle

1991 ◽  
Vol 261 (4) ◽  
pp. C608-C613 ◽  
Author(s):  
E. J. Henriksen
Keyword(s):  
Skeletal Muscle ◽  
Amino Acid ◽  
Amino Acid Uptake ◽  
Isobutyric Acid ◽  
Acid Uptake ◽  
Mammalian Skeletal Muscle ◽  
Phenylarsine Oxide ◽  
System A ◽  
Carrier Molecule ◽  
20 Nm

The role of vicinal sulfhydryls in the stimulation by insulin of system A amino acid uptake in mammalian skeletal muscle was investigated. Neutral amino acid uptake via system A carriers was assessed using the nonmetabolizable analogue alpha-(methylamino)isobutyric acid (MeAIB). Phenylarsine oxide (PAO), a trivalent arsenical that interacts with vicinal sulfhydryls, at 40 microM inhibited basal and insulin-stimulated (2 mU/ml) MeAIB uptake in rat epitrochlearis muscles by approximately 50% and approximately 80%, respectively. No significant changes in the ATP level or in the lactate-to-pyruvate ratio were observed. Both inhibitory effects were completely preventable by coincubation with dimercaptopropanol, a vicinal dithiol, indicating the effects were mediated specifically by interactions with vicinal sulfhydryls. Stimulation of MeAIB uptake by the insulin-mimicker vanadate (10 mM) or by insulin-like growth factor I (IGF-I, 20 nM) was also inhibited by 80-90% by PAO. Kinetic analysis showed that PAO decreased the apparent Vmax for basal and insulin-stimulated MeAIB uptake without altering the apparent Km. MeAIB uptake already maximally stimulated by insulin was rapidly (half-time = approximately 10 min) reversed by the addition of PAO so that the rate of MeAIB uptake was the same as in muscles incubated throughout with insulin and PAO. These results implicate a major role for vicinal sulfhydryls in the stimulation by insulin of amino acid uptake via system A carriers in skeletal muscle and suggest that the site of action of PAO on this system is distal to the insulin receptor, possibly at the carrier molecule itself.

scholarly journals Internal Ribosome Entry Site-mediated Translation of a Mammalian mRNA Is Regulated by Amino Acid Availability

2000 ◽  
Vol 276 (15) ◽  
pp. 12285-12291 ◽  
Author(s):  
James Fernandez ◽  
Ibrahim Yaman ◽  
Rangnath Mishra ◽  
William C. Merrick ◽  
Martin D. Snider ◽  
...  
Keyword(s):  
Amino Acid ◽  
Expression System ◽  
Fold Increase ◽  
Essential Amino Acids ◽  
Amino Acid Starvation ◽  
Cellular Stress Response ◽  
Entry Site ◽  
Cationic Amino Acid ◽  
Protein Levels ◽  
Amino Acid Availability

The cationic amino acid transporter, Cat-1, facilitates the uptake of the essential amino acids arginine and lysine. Amino acid starvation causes accumulation and increased translation of cat-1 mRNA, resulting in a 58-fold increase in protein levels and increased arginine uptake. A bicistronic mRNA expression system was used to demonstrate the presence of an internal ribosomal entry sequence (IRES) within the 5′-untranslated region of the cat-1 mRNA. This study shows that IRES-mediated translation of the cat-1 mRNA is regulated by amino acid availability. This IRES causes an increase in translation under conditions of amino acid starvation. In contrast, cap-dependent protein synthesis is inhibited during amino acid starvation, which is well correlated with decreased phosphorylation of the cap-binding protein, eIF4E. These findings reveal a new aspect of mammalian gene expression and regulation that provides a cellular stress response; when the nutrient supply is limited, the activation of IRES-mediated translation of mammalian mRNAs results in the synthesis of proteins essential for cell survival.

scholarly journals Regulation of System B0 amino-acid-transport activity in the renal epithelial cell line NBL-1 and concomitant changes in SAAT1 hybridizing transcripts

1994 ◽  
Vol 301 (2) ◽  
pp. 399-405 ◽  
Author(s):  
S Plakidou-Dymock ◽  
M J Tanner ◽  
J D McGivan
Keyword(s):  
Amino Acid ◽  
Cell Line ◽  
Physiological Stress ◽  
Sequence Similarity ◽  
Isobutyric Acid ◽  
Amino Acid Starvation ◽  
Epithelial Cell Line ◽  
Transport Activity ◽  
Renal Epithelial Cell ◽  
Renal Epithelial Cell Line

alpha-(Methylamino)isobutyric acid (MeAIB) insensitive Na(+)-dependent alanine transport activity in the bovine kidney cell line NBL-1 was increased upon amino acid starvation (> or = 20% over control levels). When L-phenylalanine (3 mM) was included in the starvation medium the increase was further enhanced (> or = 85% over control levels). In cells grown in control medium the Vmax, for MeAIB-insensitive Na+/alanine co-transport was found to be 6.0 +/- 0.7 nmol/3 min per mg (Km 41 +/- 12 microM) and for L-phenylalanine-treated amino-acid-starved cells the Vmax. was 21 +/- 5 nmol/3 min per mg (Km 92 +/- 40 microM). The increase in Vmax. was prevented by cycloheximide. Substrate specificity analysis identified the L-phenylalanine-induced transport system as System B0. [35S]Methionine labelling of cells during the amino acid starvation/phenylalanine treatments resulted in the differential labelling of a protein of 78 kDa. Northern-blot analysis using a SAAT1-specific probe revealed the presence of a new transcript (3.2 kb) in RNA extracted from cells incubated in amino acid starvation medium with L-phenylalanine included. The present findings suggest a novel means of control for System B0 by the use of physiological stress. It is also proposed that SAAT1 and System-B0 transcripts have considerable sequence similarity.

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