The contribution of SNAT1 to system A amino acid transporter activity in human placental trophoblast

Abstract

System A-mediated amino acid transport across the placenta is important for the supply of neutral amino acids needed for fetal growth. All three system A subtypes (SNAT1, 2, and 4) are expressed in human placental trophoblast suggesting there is an important biological role for each. Placental system A activity increases as pregnancy progresses, coinciding with increased fetal nutrient demands. We have previously shown SNAT4-mediated system A activity is higher in first trimester than at term, suggesting that SNAT1 and/or SNAT2 are responsible for the increased system A activity later in gestation. However, the relative contribution of each subtype to transporter activity in trophoblast at term has yet to be evaluated. The purpose of this study was to identify the predominant subtype of system A in cytotrophoblast cells isolated from term placenta, maintained in culture for 66h, by: (1) measuring mRNA expression of the three subtypes and determining the Michaelis-Menten constants for uptake of the system A-specific substrate, (14)C-MeAIB, (2) investigating the contribution of SNAT1 to total system A activity using siRNA.

Results: mRNA expression was highest for the SNAT1 subtype of system A. Kinetic analysis of (14)C-MeAIB uptake revealed two distinct transport systems; system 1: K(m)=0.38+/-0.12mM, V(max)=27.8+/-9.0pmol/mg protein/20min, which resembles that reported for SNAT1 and SNAT2 in other cell types, and system 2: K(m)=45.4+/-25.0mM, V(max)=1190+/-291pmol/mg protein/20min, which potentially represents SNAT4. Successful knockdown of SNAT1 mRNA using target-specific siRNA significantly reduced system A activity (median 75% knockdown, n=7).

Conclusion: These data enhance our limited understanding of the relative importance of the system A subtypes for amino acid transport in human placental trophoblast by demonstrating that SNAT1 is a key contributor to system A activity at term.

Fig. 1

(A) Box and whisker plots to show SLC38A1 and A2 mRNA expression in term cytotrophoblast cells relative to β-actin mRNA expression (n = 7). The box denotes the interval between the 25th and 75th percentile, the whiskers represent the range, and the line inside the box shows the median; ∗p < 0.01, Wilcoxon-signed rank test. (B) Kinetic modelling of ¹⁴C-MeAIB uptake in cytotrophoblast cells, using non-linear regression analysis, with one-site (dashed line) and two-site (solid line) binding components. A two-site model was preferred (F test, p < 0.05). Data are shown as median and interquartile range (n = 5).

Fig. 2

(A) SLC38A1, (B) SLC38A2, and (C) β-actin mRNA expression in cells transfected with non-targeting (NT) or SLC38A1-specfic (SNAT1) siRNA relative to corresponding mock transfected cytotrophoblast cells (dashed line at 100%). Median and interquartile range; ∗∗p < 0.01 vs 100%, Wilcoxon-signed rank test.

Fig. 3

System A activity, measured as Na⁺-dependent ¹⁴C-MeAIB uptake, by mock transfected cytotrophoblast cells and cells transfected with non-targeting (NT) or SLC38A1-specific (SNAT1) siRNA. System A activity is expressed relative to the corresponding untransfected control (dashed line at 100%). Median and interquartile range; ∗p < 0.05 vs 100%, Wilcoxon-signed rank test.