Regulation of amino acid transport System A in the human choriocarcinoma BeWo cell Line

H. N. Jones, C. J. Ashworth, K. R. Page, H. J. McArdle

Research output: Contribution to journalAbstract

Abstract

Adaptive regulation of amino acid transport system A is the response to the reduction of substrate availability seen in several cell types including adipocytes (Hyde et al. 2001). Chronic depletion of amino acids results in increased system A gene expression and de novo synthesis of new transporter proteins. We have studied the response of two system A transporters, ATA1 and ATA2, to amino acid deprivation in the human choriocarcinoma cell line BeWo. The cells were maintained in DMEM supplemented with 10 % fetal calf serum and 2 % penicillin/ streptomycin. Polycarbonate filters were seeded and cells grown to form a confluent monolayer. Amino acid deprivation was induced by culturing cells in Deprivation Media 1 (DM1, Earles Balanced Salt Solution, pH7.4 supplemented with 1 X MEM essential amino acid solution without non-essential amino acids) or DM2 (DM1 with 0.5 X MEM essential amino acid solution) for 6 hours. 14C-MeAIB trans-cellular transport was studied after pre-exposure of the cells to control and DM1 media. Protein and mRNA samples were isolated from control and treated cells and the expression of system A transporters analysed by Western and Northern blotting. Significance was assessed using non-paired t tests. MeAIB trans-cellular transport was significantly (P < 0.001, n = 9) up-regulated after exposure of the BeWo cells to DM1 compared to control. ATA2 protein expression remained at control levels after exposure of the cells to DM1. However, ATA2 mRNA levels were significantly (P < 0.0001, n = 12) increased by 6 h exposure of cells to DM1 and DM2 compared to control. In contrast, ATA1 mRNA expression was decreased (P < 0.05, n = 12) by incubation of cells in DM1 and returned to control level in DM2. We could not measure ATA1 protein levels. These results suggest that ATA2 is responsible for the adaptive regulation of system A in the BeWo cell line and that ATA1 may act as a basal transporter.
Original languageEnglish
Article numberPC113
Number of pages1
JournalThe Journal of Physiology
Volume555P
Publication statusPublished - 2004

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Amino Acid Transport System A
Choriocarcinoma
Cell Line
Amino Acids
polycarbonate
Essential Amino Acids
Messenger RNA
Proteins
Streptomycin
Adipocytes
Penicillins
Northern Blotting

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Regulation of amino acid transport System A in the human choriocarcinoma BeWo cell Line. / Jones, H. N.; Ashworth, C. J.; Page, K. R.; McArdle, H. J.

In: The Journal of Physiology, Vol. 555P, PC113, 2004.

Research output: Contribution to journalAbstract

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abstract = "Adaptive regulation of amino acid transport system A is the response to the reduction of substrate availability seen in several cell types including adipocytes (Hyde et al. 2001). Chronic depletion of amino acids results in increased system A gene expression and de novo synthesis of new transporter proteins. We have studied the response of two system A transporters, ATA1 and ATA2, to amino acid deprivation in the human choriocarcinoma cell line BeWo. The cells were maintained in DMEM supplemented with 10 {\%} fetal calf serum and 2 {\%} penicillin/ streptomycin. Polycarbonate filters were seeded and cells grown to form a confluent monolayer. Amino acid deprivation was induced by culturing cells in Deprivation Media 1 (DM1, Earles Balanced Salt Solution, pH7.4 supplemented with 1 X MEM essential amino acid solution without non-essential amino acids) or DM2 (DM1 with 0.5 X MEM essential amino acid solution) for 6 hours. 14C-MeAIB trans-cellular transport was studied after pre-exposure of the cells to control and DM1 media. Protein and mRNA samples were isolated from control and treated cells and the expression of system A transporters analysed by Western and Northern blotting. Significance was assessed using non-paired t tests. MeAIB trans-cellular transport was significantly (P < 0.001, n = 9) up-regulated after exposure of the BeWo cells to DM1 compared to control. ATA2 protein expression remained at control levels after exposure of the cells to DM1. However, ATA2 mRNA levels were significantly (P < 0.0001, n = 12) increased by 6 h exposure of cells to DM1 and DM2 compared to control. In contrast, ATA1 mRNA expression was decreased (P < 0.05, n = 12) by incubation of cells in DM1 and returned to control level in DM2. We could not measure ATA1 protein levels. These results suggest that ATA2 is responsible for the adaptive regulation of system A in the BeWo cell line and that ATA1 may act as a basal transporter.",
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N2 - Adaptive regulation of amino acid transport system A is the response to the reduction of substrate availability seen in several cell types including adipocytes (Hyde et al. 2001). Chronic depletion of amino acids results in increased system A gene expression and de novo synthesis of new transporter proteins. We have studied the response of two system A transporters, ATA1 and ATA2, to amino acid deprivation in the human choriocarcinoma cell line BeWo. The cells were maintained in DMEM supplemented with 10 % fetal calf serum and 2 % penicillin/ streptomycin. Polycarbonate filters were seeded and cells grown to form a confluent monolayer. Amino acid deprivation was induced by culturing cells in Deprivation Media 1 (DM1, Earles Balanced Salt Solution, pH7.4 supplemented with 1 X MEM essential amino acid solution without non-essential amino acids) or DM2 (DM1 with 0.5 X MEM essential amino acid solution) for 6 hours. 14C-MeAIB trans-cellular transport was studied after pre-exposure of the cells to control and DM1 media. Protein and mRNA samples were isolated from control and treated cells and the expression of system A transporters analysed by Western and Northern blotting. Significance was assessed using non-paired t tests. MeAIB trans-cellular transport was significantly (P < 0.001, n = 9) up-regulated after exposure of the BeWo cells to DM1 compared to control. ATA2 protein expression remained at control levels after exposure of the cells to DM1. However, ATA2 mRNA levels were significantly (P < 0.0001, n = 12) increased by 6 h exposure of cells to DM1 and DM2 compared to control. In contrast, ATA1 mRNA expression was decreased (P < 0.05, n = 12) by incubation of cells in DM1 and returned to control level in DM2. We could not measure ATA1 protein levels. These results suggest that ATA2 is responsible for the adaptive regulation of system A in the BeWo cell line and that ATA1 may act as a basal transporter.

AB - Adaptive regulation of amino acid transport system A is the response to the reduction of substrate availability seen in several cell types including adipocytes (Hyde et al. 2001). Chronic depletion of amino acids results in increased system A gene expression and de novo synthesis of new transporter proteins. We have studied the response of two system A transporters, ATA1 and ATA2, to amino acid deprivation in the human choriocarcinoma cell line BeWo. The cells were maintained in DMEM supplemented with 10 % fetal calf serum and 2 % penicillin/ streptomycin. Polycarbonate filters were seeded and cells grown to form a confluent monolayer. Amino acid deprivation was induced by culturing cells in Deprivation Media 1 (DM1, Earles Balanced Salt Solution, pH7.4 supplemented with 1 X MEM essential amino acid solution without non-essential amino acids) or DM2 (DM1 with 0.5 X MEM essential amino acid solution) for 6 hours. 14C-MeAIB trans-cellular transport was studied after pre-exposure of the cells to control and DM1 media. Protein and mRNA samples were isolated from control and treated cells and the expression of system A transporters analysed by Western and Northern blotting. Significance was assessed using non-paired t tests. MeAIB trans-cellular transport was significantly (P < 0.001, n = 9) up-regulated after exposure of the BeWo cells to DM1 compared to control. ATA2 protein expression remained at control levels after exposure of the cells to DM1. However, ATA2 mRNA levels were significantly (P < 0.0001, n = 12) increased by 6 h exposure of cells to DM1 and DM2 compared to control. In contrast, ATA1 mRNA expression was decreased (P < 0.05, n = 12) by incubation of cells in DM1 and returned to control level in DM2. We could not measure ATA1 protein levels. These results suggest that ATA2 is responsible for the adaptive regulation of system A in the BeWo cell line and that ATA1 may act as a basal transporter.

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VL - 555P

JO - The Journal of Physiology

JF - The Journal of Physiology

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